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POLYMERIZED BOVINE HEMOGLOBIN CAN IMPROVE SMALL-VOLUME RESUSCITATION FROM HEMORRHAGIC SHOCK IN HAMSTERS

Cabrales, Pedro*; Tsai, Amy G.*†; Intaglietta, Marcos

doi: 10.1097/SHK.0b013e318180ff63
Basic Science Aspects
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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.

*La Jolla Bioengineering Institute; and Department of Bioengineering, University of California, San Diego, La Jolla, California

Received 19 Dec 2007; first review completed 9 Jan 2008; accepted in final form 19 May 2008

Address reprint requests to Pedro Cabrales, PhD, La Jolla Bioengineering Institute, 505 Coast Blvd South Suite No. 405, La Jolla, CA 92037. E-mail: pcabrales@ucsd.edu.

This study was supported by the Bioengineering Research Partnership (grant no. R24-HL64395, grant no. R01-HL62354, grant no. R01-HL62318, and grant no. R01-HL76182).

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INTRODUCTION

The major cause of combat death is hemorrhage after penetrating trauma. Approximately 20% of battlefield casualties are potentially salvageable, and logistic problems associated with combat conditions may preclude resuscitation efforts (1). Conventional management of penetrating trauma based on Advanced Trauma Life Support guidelines has relied on aggressive fluid resuscitation until definitive control of hemorrhage has been achieved (2). Rapid recovery of intravascular and perivascular volume to restore blood pressure and metabolic imbalance is a resuscitation strategy that restores circulatory function and corrects metabolic acidosis associated with hypoperfusion and shock (3, 4). This approach was developed in part during the Vietnam War, when fluid resuscitation implemented with red blood cells (RBCs) and crystalloid solutions allowed patients who would have relented to hemorrhagic shock to survive (5). The consequences of fluid overload in trauma influenced the length of recovery, days requiring mechanical ventilation, and mortality, suggesting the need for reevaluating the methods of fluid resuscitation (6,7). In addition, aggressive attempts to normalize pressure with large amounts of fluids result in increased bleeding, hemodynamic decompensation, and mortality (7).

Red blood cell transfusion is performed after resuscitation to augment oxygen-carrying capacity in situations of high risk for inadequate oxygen delivery (8). The "transfusion trigger" or minimal hematocrit (Hct) or hemoglobin (Hb) level at which transfusion is required is subject of controversy. In some situations, the concept of a transfusion trigger has been replaced by a functional parameter by advice careful monitoring for evidence of inadequate tissue oxygen delivery on a case-by-case basis. The American Society of Anesthesiologists recommends that RBCs should be administered when Hb concentration is less than 6 g/dL in young patients (8).

Oxygen-carrying blood substitutes presently under development are proposed to improve oxygen-carrying capacity without problems of storage, compatibility, and disease transmission. Hemoglobin-based oxygen carriers (HBOCs) can be formulated with an oxygen-carrying capacity similar to blood. The HBOC solutions are the currently prevailing configuration HBOCs in clinical trials. Oxygen extraction from blood RBCs is facilitated by the presence of HBOCs because HBOCs can bring a higher concentration of oxygen bound to Hb next to vessel wall. In addition, facilitated diffusion further favors oxygen unloading from the blood column because oxygen is not only delivered by the oxygen concentration gradient, but also by the Oxyhemoglobin (OxyHb) concentration gradient (9).

Restoration of oxygen delivery is a goal of resuscitation, and it is accomplished by the restoration of microvascular perfusion and limited restoration of oxygen-carrying capacity (10). Limited reoxygenation is a factor in controlling damage during shock and for preventing further injury during the resuscitation phase. Thus, resuscitation fluids need to recover perfusion, allowing the remaining RBCs to deliver oxygen. If oxygen delivery is insufficient, the oxygen-carrying capacity can be increased with additional RBCs, or eventually, HBOCs. A strategy using low-volume resuscitation and limited oxygen-carrying capacity recovery may provide a way to prevent and control multiorgan failure from becoming established (10).

The current model corresponds to a venue where basic homeostatic conditions are recovered before prehospital settings are available by reason of entrapment, military conflicts, or other situations that prevent transport. It can also be translated to clinical situations were rapid intravascular volume replacement to restore and maintain adequate tissue perfusion may lead to increased blood loss, rapid hemodynamic deterioration, and increased mortality before the patient can be definitively treated in the operating room. The study objective was to identify the role of increasing plasma oxygen-carrying capacity for restoration of systemic and microvascular parameters from hemorrhagic shock, using a moderate-volume resuscitation strategy. To achieve this objective, we subjected our experimental hamster model to hemorrhage of 50% of blood volume (BV), continued by hypovolemic shock for 60 min. Resuscitation was implemented in two steps: the initial phase was hypertonic saline (HTS, 7.5% NaCl) 3.5% of BV, and 5 min after HTS, 10% of BV of different concentrations of Oxyglobin (approved in the United States and European Union for veterinary use, Biopure Corporation, Cambridge, Mass) was provided. Colloidal osmotic pressure (COP) of PBH solutions was balanced with human serum albumin diluted in isotonic sodium chloride solution. These findings were compared with volume resuscitation with human albumin solution at matching COP (11).

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METHODS

Animal preparation

Investigations were performed in 55- to 65-g male golden Syrian hamsters (Charles River Laboratories, Boston, Mass) fitted with a dorsal window chamber. Animal handling and care followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The experimental protocol was approved by the local animal care committee. The hamster window chamber model is widely used for microvascular studies in the unanesthetized state, and the complete surgical technique is described in detail elsewhere (12, 13). Catheters were tunneled under the skin, exteriorized at the dorsal side of the neck, and securely attached to the window frame. The microvasculature studies were performed 2 days after catheter implantation surgery.

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Inclusion criteria

Animals were suitable for the experiments if: (a) systemic parameters were within reference range, namely, heart rate (HR) greater than 340 beats/min, MAP greater than 80 mmHg, systemic Hct greater than 45%, and arterial oxygen partial pressure (PaO2) greater than 50 mmHg; and (b) microscopic examination of the tissue in the chamber observed under 650 times magnification did not reveal signs of edema or bleeding. Hamsters are a fossorial species with a lower PaO2 than other rodents because of their adaptation to the subterranean environment. However, microvascular PO2 distribution in the chamber window model is similar to other rodents, such as mice (14).

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Systemic parameters

The MAP and HR were recorded continuously (MP 150, Biopac System; Santa Barbara, Calif). The Hct was measured from centrifuged arterial blood samples taken in heparinized capillary tubes. The Hb content was determined spectrophotometrically (B-Hemoglobin, Hemocue, Stockholm, Sweden).

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Blood chemistry and biophysical properties

Arterial blood was collected in heparinized glass capillaries (0.05 mL) and immediately analyzed for PaO2, PaCO2, base excess, and pH (Blood Chemistry Analyzer 248, Bayer, Norwood, Mass). Viscosity was measured at a shear rate of 160/s (Brookfield Engineering Laboratories, Middleboro, Mass). Colloid osmotic pressure was measured using a 4420 Colloid Osmometer (Wescor, Logan, Utah).

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Limited oxygen-carrying capacity restoration

Limited restoration of oxygen-carrying capacity was attained by the infusion of the molecular Hb HBOCs Oxyglobin, a polymerized bovine Hb (PBH) material, produced by nonspecific polymerization with glutaraldehyde (mean molecular weight, 200 kd) in a modified Ringer's lactate solution. It is a distribution of Hb polymers with less than 5% of the Hb as unstabilized tetramers, approximately 50% has a molecular weight between 65 and 130 kd, and no more than 10% has a molecular weight greater than 500 kd. It also contains less than 0.1 μg/mL of free glutaraldehyde and 0.05 EU/mL endotoxin. The PBH concentrations tested were 13 g/dL and 4 g/dL.

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Acute hemorrhage and volume replacement protocol

Acute hemorrhage was induced by withdrawing 50% of estimated BV via the carotid artery catheter within 5 min (approximate mean withdrawal rate, 0.45 mL/min). Total BV was estimated as 7% of body weight. One hour after hemorrhage induction, animals received a single bolus infusion of HTS 3.5% of BV, and 5 min after HTS, 10% of BV of resuscitation fluids was infused within 10 min via the jugular vein catheter. Restoration of 13.5% of the BV does not cause reinstating normovolemia. Parameters were analyzed before hemorrhage (baseline), after hemorrhage (shock, 50 min after hemorrhage induction), and up to 90 min after volume replacement (resuscitation).

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Experimental groups

Animals were randomly divided into three experimental groups before the experiment, according to a sorting scheme based on a list of random numbers (15), namely, (a) PBH13, HTS infusion followed by volume resuscitation performed with Oxyglobin (13 g/dL; viscosity, 1.9 centipoise [cP]); (b) PBH4, HTS infusion followed by volume resuscitation Oxyglobin diluted to 4 g/dL in albumin solution; (c) PBH0, HTS infusion followed by volume resuscitation with albumin solution. The COP of all solutions was 40 mmHg. Viscosities of 4% of PBH and albumin solution were 1.3 cP and 1.2 cP, respectively. Eighteen animals were entered into the study, and all tolerated the entire protocol without visible signs of discomfort. Animals were randomly assigned to the following experimental groups: PBH13 (n = 6), PBH4 (n = 6), and PBH0 (n = 6). Systemic data for baseline and shock were obtained by combining all experimental groups. Six additional animals were used to determine blood and plasma viscosity, and plasma COP.

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Microvascular experimental setup

The unanesthetized animal was placed in a restraining tube with a longitudinal slit from which the window chamber protruded, then fixed to the microscopic stage of a transillumination intravital microscope (BX51WI, Olympus, New Hyde Park, NY). The animals were given 20 min to adjust to the change in the tube environment before measurements. Measurements were carried out using a 40 times (LUMPFL-WIR, numerical aperture 0.8, Olympus) water immersion objective.

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Functional capillary density

Functional capillaries, defined as those capillary segments that have RBC transit of at least one RBC in a 60-s period in 10 successive microscopic fields were assessed, totaling a region of 0.46 mm2. Each field had 2 to 5 capillary segments with RBC flow. The functional capillary density ([FCD] per centimeter), that is, total length of RBC perfused capillaries divided by the area of the microscopic field of view, was evaluated by measuring and adding the length of capillaries that had RBC transit in the field of view. The relative change in FCD from baseline levels after each intervention is indicative of the extent of capillary perfusion (16, 17).

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Hemoglobin oxygen saturation

Oxygen saturation curves for RBCs were obtained by deoxygenation of O2-equilibrated samples in the Hemox buffer at 37.6°C, using a Hemox Analyzer (TCS Scientific Corporation, New Hope, Pa) (11).

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Microhemodynamics

Arteriolar and venular blood flow velocities were measured online by using the photodiode cross-correlation method (Photo-Diode/Velocity, Vista Electronics, San Diego, Calif) (18). The measured centerline velocity (V) was corrected according to vessel size to obtain the mean RBC velocity (19). A video image-shearing method was used to measure vessel diameter (D) (20). Blood flow (Q) was calculated from the measured values as Q = π × V (D/2)2. Peripheral vascular resistance was calculated as the ration of normalized to baseline MAP against normalized arteriolar blood flow. Peripheral vascular hindrance was calculated as the ratio of peripheral vascular resistance against the normalized to baseline blood viscosity. Wall shear stress (WSS) was defined by WSS = WSR × η, where WSR is the wall shear rate given by 8VD−1, and η is the microvascular blood viscosity or plasma viscosity.

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Microvascular PO2 distribution

High-resolution noninvasive microvascular PO2 measurements were made using phosphorescence quenching microscopy (PQM) (21, 22). The PQM is based on the O2-dependent quenching of phosphorescence emitted by albumin-bound metalloporphyrin complex after pulsed light excitation. The PQM is independent of the dye concentration within the tissue and is well suited for detecting hypoxia because its decay time is inversely proportional to the PO2 level, causing the method to be more precise at low PO2. This technique is used to measure both intravascular and extravascular PO2 because the albumin-dye complex continuously extravasates from the circulation into the interstitial tissue (21, 22). Tissue PO2 was measured in tissue regions in-between functional capillaries. The PQM allows for precise localization of the PO2 measurements without subjecting the tissue to injury. These measurements provide a detailed understanding of microvascular O2 distribution and indicate whether O2 is delivered to the interstitial areas.

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Oxygen delivery and extraction

The microvascular methodology used in our studies allows a detailed analysis of O2 delivery to the tissue. Calculations are made using equations 1, O2 delivery (DO2), and 2, O2 extraction (VO2) (16).

where RBCHb is the Hb from RBCs, Total Hb - plasma Hb (gHb/dLblood), PlasmaHb is the acellular Hb (gHb/dLblood), γ is the O2-carrying capacity of saturated Hb (1.34 mL O2/gHb), SA% is the arteriolar blood O2 saturation, SA% is the arteriolar PBH O2 saturation, A−V indicates the arteriolar/venular differences, and Q is the microvascular flow. The O2 saturations were measured as described previously.

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Data analysis

Results are presented as mean ± SD. Data within each group were analyzed using analysis of variance for repeated measurements (Kruskal-Wallis test). When appropriate, post hoc analyses were performed with the Dunn multiple comparison test. Microhemodynamic data are presented as absolute values and ratios relative to baseline values. A ratio of 1.0 signifies no change from baseline, whereas lower and higher ratios are indicative of changes proportionally lower and higher than baseline (i.e., 1.5 would mean a 50% increase from the baseline level). The same vessels and capillary fields were followed so that direct comparisons to their baseline levels could be performed, allowing for more robust statistics for small sample populations. All statistics were calculated using GraphPad Prism 4.01 (GraphPad Software, Inc, San Diego, Calif). Changes were considered statistically significant if P < 0.05.

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RESULTS

Systemic parameters

Systemic hemodynamic and blood parameters are presented in detail in Table 1. Hemorrhage statistically reduced Hct and Hb from baseline. The PBH13, PBH4, and PBH0 resuscitation decreased Hct and total Hb. Prolonged volume expansion was observed up to 90 min after resuscitation; at this point, Hct and total Hb for PBH13, PBH4, and PBH0 were lower than baseline and shock. The hamster hemorrhage model has low variability as noted by the consistency in the results observed during shock.

Table 1

Table 1

Changes in MAP during the hemorrhagic shock resuscitation protocol for all experimental groups are presented in Table 1. Hemorrhage and shock statistically decreased MAP from baseline. The PBH13 resuscitation restored MAP to baseline at 60 and 90 min after resuscitation. The PBH4 resuscitation restored MAP from shock, although statistically lower than baseline at all time points. The MAP was different between PBH13 and PBH4 at all time points after resuscitation. The PBH0 resuscitation moderately recovered MAP from shock and statistically lower than PBH13 and PBH4. Heart rates were not different among groups after resuscitation.

Gas laboratory parameters and calculated acid-base balance are presented in Table 1. Hemorrhagic shock significantly decreased arterial pH and PCO2, compromising acid-base balance. The PBH13 resuscitation partially recovered blood gas parameters; however, acid-base balance remained negative. The PBH4 resuscitation recovered blood pH and restored a positive acid-base balance up to 90 min. The PBH0 resuscitation did not reverse negative acid-base balance from shock. All resuscitation strategies mitigated arterial acidosis produced during shock conditions.

The PBH13, PBH4, or PBH0 resuscitation did not change plasma viscosity. Blood viscosity and plasma COP were statistically reduced after resuscitation for all groups (compared with normal blood viscosity, 4.2 ± 0.1 cP). Table 2 shows the blood rheological properties and COP for normal blood and after resuscitation. Baseline rheological properties and COP were obtained from blood of hamsters that did not undergo the shock protocol.

Table 2

Table 2

Microvascular diameter and blood flow are presented in Figure 1. Arteriolar and venular diameters were significantly constricted from baseline during shock. The PBH0 resuscitation reversed vasoconstriction. The PBH13 and PBH4 resuscitation remained vasoconstricted after resuscitation. Venular diameters 60 min after resuscitation recovered with PBH4 and after 90 min also for PBH0. The PBH13 did not restore venular diameters after resuscitation.

Fig. 1

Fig. 1

Arteriolar and venular flows during shock were statistically significantly lower than baseline for all groups. Resuscitation increased microvascular flows in all groups. The PBH4 presented statistically higher arteriolar and venular blood flows than PBH13 and PBH0. The PBH13 had statistically lower arteriolar and venular flows than PBH0.

Changes in capillary perfusion during the protocol are presented in Figure 2. The FCD was significantly reduced after hemorrhage and shock. Resuscitation partially restored FCD in all groups. Sixty minutes after resuscitation, PBH4 had higher FCD than PBH13 and PBH0. The FCD for PBH13 was lower than PBH0. Ninety minutes after resuscitation, differences were smaller among groups, and PBH4 maintained significantly higher levels of FCD when compared with PBH13 or PBH0.

Fig. 2

Fig. 2

Calculated hemodynamic parameters are presented in Table 2. Peripheral vascular resistance was statically increased during shock. The PBH13 resuscitation had higher vascular resistance than PBH4 or PBH0. Peripheral vascular hindrance, which reflects the contribution of vascular geometry, presented an identical trend as the vascular resistance because of similarities in blood viscosity among groups. The PBH4 had lower vascular resistance and higher levels of vessel wall shear rate and stress at the microcirculation when compared with those of PBH13 and PBH0.

Intravascular and tissue O2 tensions 90 min after resuscitation are shown in Figure 3. The PBH4 yielded the highest arteriolar, venular, and tissue PO2 when compared with PBH13 and PBH0. There were no differences in intravascular and tissue PO2 between PBH13 and PBH0. Figure 4A shows the result of the analysis of oxygen delivery and extraction 90 min after resuscitation. The PBH13 had higher systemic oxygen delivery when compared with those of PBH4 and PBH0 because of the higher plasma oxygen-carrying capacity. Systemic (arterial to arteriolar; premicrovascular) oxygen extraction was lower for PBH4 when compared with PBH13 and PBH0. Resulting from the lower premicrovascular oxygen release, PBH4 produced a higher microvascular oxygen delivery and extraction than PBH13 and PBH0. Figure 4B shows the extraction ratio (delivery over extraction) from RBCs and plasma Hb in all groups. The PBH4 reduced the amount of oxygen extracted from RBCs, as the plasma Hb increased the amount supplied by RBC (Fig. 4A, arteriolar delivery). Figure 4C shows the microvascular oxygen reserve (difference delivery and extraction normalized by extraction) from RBC and plasma Hb in all groups. The PBH4 increased oxygen reserve in RBCs and plasma Hb when compared with PBH13 and PBH0.

Fig. 3

Fig. 3

Fig. 4

Fig. 4

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DISCUSSION

The principal finding of this study is that small-volume resuscitation from hemorrhagic shock by infusion of HTS followed by a moderated concentration of HBOC (PBH4) provides superior restoration of systemic and microhemodynamic parameters when compared with a greater amount of HBOC (PBH13) or in the absence of HBOC (PBH0). The importance of increasing plasma oxygen-carrying capacity during anemic and hypovolemic conditions is shown by the sustained recovery of microhemodynamic conditions postresuscitation when PHB is added to the circulating blood at different concentrations (PBH13 and PBH4). Colloidal osmotic properties were not a factor during the study because solution COP was matched before infusion, verified by the lack of differences in Hct between groups. Therefore, because there were no differences in RBC-based oxygen transport capacity (Hct), the differences found between groups should mostly be caused by the changes in plasma oxygen-carrying capacity by the PBH solution (Oxyglobin). It should be noted that awake animals respond differently from anesthetized animals; therefore, these results can be more strongly extrapolated to the treatment of injured subjects.

Resuscitation with only HTS partially restores blood pressure and microhemodynamics. Our previous results suggest that small-volume resuscitation accentuates fluids shifts, even though it does not stabilize them (23, 24). Furthermore, the addition of a hyperoncotic solution after HTS sustains and improves systemic and hemodynamic parameters, although increasing the anemic state (23, 24). The systemic recovery in MAP observed after infusion of PBH after HTS was proportional to the concentration of the infused Hb in plasma. Our results confirm that small-volume (HTS) resuscitation can ensure adequate hemodynamic restoration when enhanced by moderate increases in plasma oxygen-carrying capacity with PBH. A low concentration of PHB recovered microvascular tone and perfusion. Blood flow velocities were the major determinant for the increase in perfusion between study groups, and calculated vascular resistance was lower for low-concentration PBH (PBH4). This result points out that oxygen-carrying HBOC formulations can be used to improve resuscitation from hemorrhagic shock by restoring microvascular perfusion without increasing peripheral vascular resistance.

Results on oxygen delivery, extraction, and tissue PO2 90 min after resuscitation show that perfusion can be more important than the intrinsic increase in oxygen-carrying capacity, that is, blood Hb concentration. Low-concentration PBH increased arteriolar PO2, leading to a significant recovery in tissue PO2. Perfusion is also dependant on how central blood pressure is transmitted to the capillary circulation, therefore, these results may be in part caused by PBH being vasoactive, leading to increased blood pressure. Blood pressure changes as a function of arteriolar tone in the absence of major blood rheological changes, consequently a moderate increase in blood pressure, can be beneficial if the related vasoconstriction does not produce a significant change in FCD.

Furthermore, our microvascular oxygen transport results show a small amount of PBH added to the blood during small-volume resuscitation is beneficial. In the absence of PBH in plasma, blood RBCs released the same quantity of oxygen before arriving to the microcirculation and at the microcirculation (50% before and 50% in the microcirculation, premicrocirculation and microcirculatory extraction). This distribution was somewhat changed by the presence of 1.6 gPBH/dL in plasma (PBH13) to RBCs releasing 41% before the microcirculation and 46% in the microcirculation, the remainder 13% of the total oxygen was offloaded from plasma Hb, divided in 7% before and 6% in the microcirculation. The presence of 0.6 gPBH/dL in plasma (PBH4) favored the release of oxygen from RBC in the microcirculation, dividing the total oxygen extracted in 35% before and 65% in the microcirculation. The lower concentration of PBH in plasma mostly affected the release of oxygen from RBCs, where 32% and 62% of total oxygen was released before and in the microcirculation, respectively. The 0.6 gPBH/dL in plasma provided even amounts of oxygen before (3% of the total oxygen) and in the microcirculation.

Moderated supplementation of oxygen-carrying capacity by PBH is more effective than a large increase because oxygen was prevalently released in the microcirculation, decreasing precapillary oxygen offload, probably avoiding triggering vascular oxygen delivery regulatory mechanisms (9). The amount of oxygen transported is determined by Hct, oxygen affinity, and arterial PO2 (10). Under normal physiological conditions, only about 25% of the oxygenated Hb in the RBC will be deoxygenated, whereas 75% recirculates, returning to the lungs (25). Thus, a major portion of the oxygen present in blood is not metabolically active. After hypovolemic resuscitation, restoration of perfusion and oxygenation are desirable. However, if a plasma expander (colloid or crystalloid) is used, only perfusion can be corrected and not intrinsic oxygen-carrying capacity, rendering restoration of blood Hb concentration necessary. Our findings suggest that even though HBOCs have been proposed for solving this problem without success, because of the inherent vasoconstrictive properties of the materials, this problem can be in part circumvented by simple titration of the amount of HBOC used during resuscitation.

The calculated oxygen extraction ratios (oxygen extracted over oxygen delivered) show that in the absence of acellular Hb, 95% of the total oxygen is extracted. When 1.6 gPBH/dL is present in plasma, 84% of the total oxygen is extracted, and if the concentration in plasma is only 0.6 gPBH/dL, the oxygen extraction ratio is decreased to 81%. Thus, small amounts of PBH in plasma not only increases the amount of oxygen delivered and extracted, it also makes the process of oxygen loading more effective, thus increasing the oxygen reserved. The addition of PBH to the resuscitation fluid increased oxygen-carrying capacity, however, precapillary oxygen is preserved, whereas microvascular oxygen delivery and extraction increased. Therefore, oxygen redistribution by addition of a moderate amount of PBH was effective in targeting oxygen release to the microcirculation.

Microvascular perfusion is governed by FCD, which is regulated by arteriolar tone (16, 26), which is controlled to a large degree by NO primarily generated by endothelial NO (27). Blood modulates NO by the joint effects of mechanotransduction, blood flow, and NO scavenging by Hb in RBCs (28). When HBOCs are infused, NO reacts rapidly with acellular OxyHb to produce methemoglobin and nitrate (28). This rapid and irreversible reaction at high concentrations of intravascular acellular OxyHb implies that the endothelium-derived NO is incapable of autocrine diffusion to smooth muscle (28-30). As observed here, if the concentration of Hb in plasma is less than 1 g/dL, the reaction between acellular OxyHb and NO seems not to completely inactivate endothelial NO. Moreover, HBOC-inherent vasoconstriction presumably caused by NO scavenging does not correlate with HBOC NO-binding properties, which can be contradictory (30, 31) because all HBOC formulations, whether vasoconstrictive or not vasoconstrictive, seem to affect perivascular NO (32). Thus, vasoconstriction cannot solely be explained by NO scavenging.

Two complementary hypotheses for HBOC vasoactivity have been proposed: (a) large Hb molecules have slow diffusion properties when compared with small Hb molecules, and therefore do not overload arterioles with oxygen (a cause of vasoconstriction because of metabolic autoregulation); and (b) dynamically large Hb molecules are not effective NO scavengers because they cannot extravasate and intercalate between the endothelium and smooth muscle. Although vasoactivity of PBH13 could be satisfactorily explained by NO scavenging and extravasation, vasoinactivity of PBH4 cannot be easily explained. Conventional explanations about vasoactivity apply to larger concentrations of PBH used as HBOCs, but they do not explain the lack of vasoactivity of lower concentrations. On the basis of the current findings, it seems that PBH causes different degrees of vasoconstriction and hypertension proportional to the concentration in the circulation. Our results tend to support the fact that the mechanism of vasoconstriction is caused by oxygen autoregulation because vasoconstriction was mostly present in the PBH13 group. This group has the largest oxygen extraction before blood arriving to the microcirculation, a signal for oxygen oversupply, activating autoregulatory mechanism to limit oxygen delivery by increasing peripheral vascular resistance, and lowering blood flow. However, it should be noted that in vivo hemodynamic responses are influenced by many factors, including BV, viscosity, ratio between acellular Hb and native RBCs, and anemic state. Briefly, PBH elicits dose-dependent vascular changes; nonetheless, when assessing Hb-mediated vasoconstriction, one should carefully define the characteristics of the Hb tested and the models used in the evaluation.

A moderate increase in plasma oxygen-carrying capacity with PBH provided better restoration of homeostasis when compared with volume resuscitation with high-concentration PBH or non-PBH. Although PBH is a broad mix of different molecular sizes, it seems that at low concentrations, the vasoconstrictive properties of the smaller Hb polymers are compensated for by the improved oxygen availability caused by facilitated oxygen diffusion and restoration of blood pressure. The net positive effect therefore seems to be caused by the balance between positive (e.g., increased oxygen-carrying capacity and perfusion pressure) and negative (e.g., vasoconstriction) stimuli.

The oxygen-binding properties of the PHB solution are the result of nonspecific polymerization with glutaraldehyde, which produces an HBOC with lower oxygen affinity (P50, 54 mmHg) than blood (normal hamster RBC P50, 32 mmHg) and reduced cooperativity, producing a material that easily releases oxygen (33). This facilitated oxygen release has been interpreted as a negative property of HBOCs when used to fully replace or reinstate blood oxygen-carrying capacity and, in combination with the inherent vasoactivity of acellular Hb, limit their use. To date, it is not possible to fully replace RBC oxygen transport properties, however, enhancement of oxygen transport and offload from the remaining RBCs by small additions of acellular Hb solution could be an important alternative.

The potential of HBOCs to enhance perfusion and oxygenation in the prehospital and combat settings has been tested without physiological explanation for the findings. Gulati and Sen (34) reported the efficacy of diaspirin cross-linked Hb during resuscitation. However, human trials using the same solutions in severe traumatic hemorrhagic shock patients were stopped after preliminary analysis of results revealed a significant increase in mortality in the diaspirin cross-linked Hb-treated group (35). Two recent trials using PBH (HBOC-201; Biopure Corporation) suggest that it may be effective during limited volume resuscitation from hemorrhagic shock. McNeil et al. (36) used a controlled hemorrhage model to compare the efficacy of PBH during hypotensive resuscitation, reporting no differences in survival rates. The study by Manning et al. (37) used a model of uncontrolled liver exsanguinations injury in swine to compare resuscitation with HBOC-201 and crystalloid solution to a target MAP (60 mmHg). They reported higher survival rates and lower lactate levels in the HBOC-201-treated animals. Amounts of PBH in plasma in these studies were similar to the current study, but infused volumes were different. In addition, the material used in the current study is the veterinarian HBOC, which is different from HBOC-201, especially the molecular size distribution.

In conclusion, the criteria for an optimal amount of PBH are not unique and require differentiation of the objective between blood pressure, blood perfusion, microvascular function, and oxygen delivery. Our results show that the latter can be accomplished via the moderate increase in plasma oxygen-carrying capacity even when the molecular oxygen carrier is vasoactive. In this context, moderate amounts of PBH used during resuscitation increased microvascular PO2, oxygen delivery, and extraction, and facilitated microvascular oxygen offload from RBCs. These effects are reversed at higher concentrations of PBH, which produce an increase in peripheral vascular resistance, negating the oxygen transport benefits observed at lower concentrations. At higher PBH concentrations, the increase in vascular resistance produced an increase of blood pressure, which may be the combined effect of inherent HBOC NO scavenging and the enhanced premicrocirculatory oxygen release. The optimal benefit of PBH seems to be limited to the appropriated concentrations, depending on the oxygen properties of the Hb solution and the remaining level of native RBC. These results suggest that there may be an upper limit for increasing acellular Hb oxygen-carrying capacity.

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ACKNOWLEDGMENTS

The authors thank Froilan P. Barra and Cynthia Walser for the surgical preparation of the animals.

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Keywords:

Microcirculation; hemorrhage; resuscitation; blood substitutes; intravascular oxygen; functional capillary density; vasoactivity; oxygen autoregulation

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