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Am J Physiol Heart Circ Physiol 288: H2146-H2153, 2005. First published January 6, 2005; doi:10.1152/ajpheart.00749.2004
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Effects of extreme hemodilution with hemoglobin-based O2 carriers on microvascular pressure

Pedro Cabrales, Amy G. Tsai, Robert M. Winslow, and Marcos Intaglietta

Department of Bioengineering, University of California-San Diego, La Jolla, California

Submitted 27 July 2004 ; accepted in final form 30 December 2004


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

shear stress; perfusion; plasma expander; blood pressure; blood substitutes

THE DEVELOPMENT of hemoglobin-based oxygen-carrying plasma expanders (OCPEs) comprises a variety of chemical modifications of the hemoglobin molecule aimed at reducing its toxicity and prolonging retention time. The goal for these products is that they can be used instead of blood upon reaching the transfusion trigger [the hematocrit (Hct)/hemoglobin concentration at which the decision is made to restore loss of the oxygen carrying capacity of blood]. Currently, these fluids include hemoglobin molecular solutions and suspensions of liposome-encapsulated hemoglobin (21, 32, 33). Molecular formulations of OCPEs have been tested in clinical trials, having mostly met with unfortunate failures that have been principally attributed to their vasoactivity. Vasoactivity of modified hemoglobin solutions is attributed to nitric oxide (NO) scavenging (7), oxygen autoregulation due to facilitated diffusion (19), and a direct pharmacological effect on smooth muscle mediated by formation of oxygen free radicals formed as the molecule extravasates and releases hemes in the interstitium (1).

An important consequence of vasoactivity is the decrease of tissue perfusion, capillary pressure, and, consequently, functional capillary density (FCD). FCD is a parameter that has been shown to be critical for tissue survival in hemorrhagic shock (15) and to be directly related to the maintenance of global homoeostatic conditions such as acid-base balance (27). Maintenance of adequate levels of FCD in extreme hemodilution was recently demonstrated to be directly related to the maintenance of a threshold level of capillary pressure, a process that in the study of Cabrales et al. (4) was achieved by elevating plasma viscosity using Dextran 500. In these experiments, maintenance of capillary pressure was attributed to a redistribution of hydraulic pressure losses. When the overall central circulation blood viscosity is diminished by a decrease of red blood cell (RBC) concentration, the improved plasma viscosity increases microcirculation apparent viscosity, a region where Hct is not as significant a determinant of viscous losses as in the central circulation (17).

Introduction of OCPEs in the circulation upon reaching the Hct reduction of transfusion trigger produces a condition of extreme hemodilution, where the oxygen-carrying capacity of OCPEs is emphasize. OCPEs such as {alpha}{alpha}-cross-linked hemoglobin ({alpha}{alpha}Hb) and polymerize bovine hemoglobin (PBH, Oxyglobin, Biopure; Cambridge, MA) have viscosities that are similar to plasma. Therefore, upon introduction into the circulation, they lead to a low-plasma viscosity extreme hemodilution condition, which was shown to reduce FCD in the study of Cabrales et al. (3). In addition, {alpha}{alpha}Hb and PBH are vasoactive (10, 11, 28) and superposed to the effects due to low plasma viscosity create an additional obstacle to the transmission of central blood pressure to the periphery.

MP4 (MAL-Peg hemoglobin, 4.2%, Sangart; San Diego, CA) is an oxygen-carrying molecule with comparatively large dimensions, relatively high viscosity, and high oxygen affinity and oncotic pressure. The study by Tsai et al. (28) showed that the presence of reduced amounts of this material in blood (1.0 g hemoglobin/dl), compared with similar studies using PBH, maintained oxygen delivery at a significantly reduced hemoglobin concentration while not significantly affecting plasma viscosity. This study also showed that FCD was statistically significantly lower using PBH than when using MP4 (36.7% PBH vs. 66.5% MP4, P < 0.05), whereas mean arteriolar and venular diameters were statistically unchanged from baseline. Arteriolar and venular flows were statistically the same as baseline for MP4, whereas they were statistically lower for PBH (P < 0.05). Changes in arteriolar diameter may be too small and variable to achieve statistical significance in this type of investigation, particularly in an awake model; however, FCD is a sensitive measurement of vasoactivity, as shown by Lindbom and Arfors (16). A possible explanation for this effect is that MP4 is vasoinactive, thus allowing for a greater portion of central blood pressure to be transmitted to the capillaries compared with a vasoactive material such as PBH.

The present study was carried out to investigate how capillary pressure and FCD are related in extreme hemodilution when oxygen-carrying capacity is partially restored using MP4 or {alpha}{alpha}Hb. MP4 was shown in previous studies to be either vasoinactive or to produce lesser levels of vasoactivity (8), whereas {alpha}{alpha}Hb is known to be vasoactive (32). Thus this study was also made to test the hypothesis that vasoconstriction reduces FCD.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
Animal preparation. Investigations were performed in 55- to 65-g golden Syrian hamsters (Charles River Laboratories; Boston, MA). Animal handling and care were provided following the procedures outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals (National Research Council, 1996). The study was approved by the local Animal Subjects Committee. The hamster window chamber model is widely used for microvascular studies in the unanesthetized state, and the complete surgical technique has been described in detail elsewhere (5, 9). Briefly, the animal was prepared for chamber implantation with a 50 mg/kg ip injection of pentobarbital sodium anesthesia. After hair removal, sutures were used to lift the dorsal skin away from the animal, and one frame of the chamber was positioned on the animal's back. A chamber consisted of two identical titanium frames with a 15-mm circular window. With the aid of backlighting and a stereomicroscope, one side of the skin fold was removed following the outline of the window until only a thin layer of retractor muscle and the intact subcutaneous skin of the opposing side remained. Saline and then a coverglass were placed on the exposed skin held in place by the other frame of the chamber. The intact skin of the other side was exposed to the ambient environment. The animal was allowed at least 2 days for recovery; its chamber was then assessed under the microscope for any signs of edema, bleeding, or unusual neovascularization. Barring these complications, the animal was anesthetized again with pentobarbital sodium. Arterial and venous catheters (polyethylene-50) were implanted in the carotid artery and jugular vein. Catheters were tunneled under the skin and exteriorized at the dorsal side of the neck, where they were attached to the chamber frame with tape (4).

Inclusion criteria. Animals were suitable for the experiments if 1) systemic parameters were within normal range, namely, heart rate (HR) > 340 beats/min, mean arterial pressure (MAP) > 80 mmHg, systemic Hct > 45%; and arterial PO2 (PaO2) > 55 mmHg; and 2) microscopic examination of the tissue in the chamber observed under x650 magnification did not reveal signs of edema or bleeding (26).

Hemoglobin solutions. MP4 is polyethylene glycol-modified human hemoglobin manufactured by Sangart (San Diego, CA), and its preparation and properties have been described elsewhere (29). {alpha}{alpha}Hb was made according to the method of Winslow and Chapman (34). Solution properties are given in Table 1.


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  		Table 1. Physical characteristics of the solutions

 
Systemic parameters. MAP, mean venular pressure (MVP; jugular catheter), and HR were recorded continuously (MP 150, Biopac Systems; Santa Barbara, CA) except during the actual blood exchange. Hct was measured from centrifuged arterial blood samples taken in heparinized capillary tubes (Readacrit Centrifuge, Clay Adams, Division of Becton-Dickinson; Parsippany, NJ). Hemoglobin content was determined spectrophotometrically from a single drop of blood (B-Hemoglobin, Hemocue; Stockholm, Sweden).

Blood chemistry and rheological properties. Arterial blood was collected in heparinized glass capillaries (0.05 ml) and immediately analyzed for PaO2, arterial PCO2 (PaCO2), base excess, and pH (Blood Chemistry Analyzer 248, Bayer; Norwood, MA). The comparatively low PaO2 and high PaCO2 of these animals is a consequence of their adaptation to a fossorial environment. Blood samples for viscosity and colloid osmotic pressure (COP) measurements were quickly withdrawn from the animal with a heparinized 5-ml syringe at the end of the experiment for immediate analysis.

Blood samples were centrifuged, and COP in the plasma was measured using a membrane colloid osmometer (model 420, Wescor; Logan, UT). Calibration of the osmometer was made with a 5% albumin solution using a 30-kDa cutoff membrane (Amicon; Danvers, MA) (30). The viscosity of plasma and whole blood was determined with a cone and plate viscometer at a shear rate of 160 s–1 at 37°C (Dv-II+ Viscometer, Brookfield Engineering Laboratories; Middleboro, MA).

FCD. Capillaries were considered functional if RBCs transited through the capillary segments during a 45-s period. FCD was tabulated from the capillary lengths with RBC transit in an area comprising 10 successive microscopic fields (420 x 320 µm2). FCD (cm–1) is the total length of RBC-perfused capillaries divided by the area of the microscopic field of view (4, 15, 26).

Microhemodynamics. Arteriolar and venular blood flow velocities were measured on-line using the photodiode cross-correlation method (12) (Photo Diode/Velocity Tracker model 102B, Vista Electronics; San Diego, CA). The measured centerline velocity (V) was corrected according to vessel size to obtain the mean RBC velocity (18). A video image-shearing method was used to measure vessel diameter (D) (13). Blood flow (Q) was calculated from the measured values as Q = V x {pi}(D/2)2. Changes in arteriolar and venular diameter from baseline were used as indicators of a change in vascular tone. Wall shear stress (WSS) was defined by WSS = WSR x {eta}, where WSR is the wall shear rate given by 8VD–1 and {eta} is blood viscosity (4, 26).

In conditions of extreme hemodilution with Hct ~11%, the contribution of RBCs to the total viscosity of blood is approximately linear, with RBCs accounting for about 0.70 cP, which is the difference between blood and plasma viscosity. According to Lipowsky and Firrell (17), the ratio between arteriolar-venular and systemic Hct illustrated a tendency to equilibrium during extreme hemodilution, converging to an average value of the ratio of 0.7 between microvascular and systemic Hct (systemic Hct ~ 10%). Therefore, we corrected our extreme hemodilution viscosity data using a blood viscosity resulting from the addition of the viscosity contribution of RBCs (at 11% Hct) reduced by 30% and the value of measured plasma viscosity at Hct 11%. The same procedure was used for the normal blood data, where the Hct reduction is 0.58 for arterioles and 0.68 for venules (17). However, because at normal Hct blood viscosity is not linearly proportional to Hct, we used actual viscosity versus Hct (dilution with hamster blood) data to obtain the corrected value for blood viscosity (4).

Microvascular pressure. Microvascular pressures were measured with the servo-nulling technique developed by Wiederhielm et al. (31). In this method, the unknown intravascular pressure is compared with a known, controlled pressure generated by a voltage-to-pressure converter. The principal features of this technique have been described in detail by Intaglietta (14) and were recently reviewed by Cabrales et al. (4). Pressure is measured via 2 M NaCl-filled micropipettes controlled using a hydraulic joystick micromanipulator (MO-102, Narishige Scientific Instruments; Tokyo, Japan) adapted for use with an Olympus BX51WI microscope.

Pressure measurements were accepted only if the following criteria were satisfied: 1) the micropipette could be manipulated inside the vessel lumen without affecting the pressure tracing; 2) the pressure tracing was insensitive to small changes in the gain of the servo-null system; and 3) the pressure tracing returned to zero when the pipette was removed from the vessel but remained immersed into the superfusate (6).

Acute isovolemic hemodilution. Progressive hemodilution to a final systemic Hct level of 25% of baseline was accomplished with three isovolemic exchange steps. This protocol has been described in detail in our previous report (24). Briefly, the volume of each exchange-transfusion step was calculated as a percentage of the blood volume, estimated as 7% of the body weight. An acute anemic state was induced by lowering systemic Hct by 60% with two steps of progressive isovolemic hemodilution using 6% Dextran 70, referred to as exchange levels 1 and 2. Level 1 exchange was 40% of blood volume, and level 2 and 3 exchanges were each 35% of blood volume.

After the level 2 exchange, level 3 exchange was performed with animals randomly divided into two experimental groups by sorting a set of random numbers produced in a random ordering scheme (2). Experimental group 1, labeled {alpha}{alpha}Hb, was hemodiluted with {alpha}{alpha}Hb to a Hct of 11%. Group 2, labeled MP4, was similarly hemodiluted with MP4. This procedure was used to compare with previous studies of extreme hemodilution using OCPEs and oxygen noncarrying plasma expanders (2–4, 26). Results were compared with control (no hemodilution) and to a previous study carried out under identical circumstances using PBH solution (13.1 g hemoglobin/dl) in a modified lactated Ringer solution (Oxyglobin, Biopure; Boston, MA) (3, 23).

Because mixed blood is withdrawn during the exchanges, a 110% blood volume exchange was needed to reduce the Hct by 75% of baseline (Hct ~11%). Solutions were infused into the jugular vein catheter after passing through an in-line, 13-mm-diameter, 0.2-µm syringe filter at a rate of 100 µl/min. Blood was simultaneously withdrawn by a dual syringe pump (33 syringe pump, Harvard Apparatus; Holliston, MA) at the same (isovolemic-normovolemic) rate from the carotid artery catheter (4, 24, 26). This slow rate of exchange provided for a stable MAP immediately after the exchange. The animal was allowed a 10-min stabilization period before data acquisition.

Experimental setup. The unanesthetized animals were placed in a restraining tube with a longitudinal slit from which the window chamber projected outward. The animals were given 30 min to adjust to the tube environment before the control systemic parameters (MAP, HR, blood gases, and Hct) were measured. The conscious animal in the tube was then affixed to the microscopic stage of a transillumination intravital microscope (BX51WI, Olympus; New Hyde Park, NY). The tissue image was projected onto a charge-coupled device camera (COHU 4815) connected to a videocassette recorder (AG-7355, JVC) and viewed on a monitor. Measurements were carried out using a x40 (LUMPFL-WIR, numerical aperture 0.8, Olympus) water immersion objective. During micropressure measurements, observations were made with either a x10 (Leitz, numerical aperture 0.22) or a x20 (Leitz, numerical aperture 0.33) dry objective. For easier detection of RBC passage, the contrast between RBCs and tissue was enhanced with a BG12 (420 nm) bandpass filter.

Fields of observation and vessels were chosen for study at locations in the tissue where the vessels were in sharp focus. Detailed mappings were made of the chamber vasculature so that the same microvessels were studied throughout the experiment. Blood samples were withdrawn from level 3 exchange animals at the end of the experiment for subsequent analysis of viscosity and COP (4, 25).

The coverglass of the window chamber was removed at the completion of the microhemodynamic measurements after the level 3 exchange, and the tissue preparation was superfused (~5 ml/min) with a physiological salt solution of the following composition (in mM): 131.9 NaCl, 4.7 KCl, 2.0 CaCl2, 1.2 MgSO4, and 20.0 NaHCO3, with pH 7.4 at 37°C. The tissue was maintained at 33–34°C by the heated solution. The solution spread on the tissue as a thin film, drained into a platter, and was drawn off by suction. Under control conditions, the solution was equilibrated with 95% N2-5% CO2, which maintained suffusate pH at 7.4 and minimized O2 delivery from the superfusate to the tissue (22, 23). Pressure measurements were initiated 20 min after glass window removal, a period that has been found to allow the tissue to stabilize and presents unchanged microvascular parameters (4).

Data analysis. Results are presented as means ± SD unless otherwise denoted; n is the number of animals and N is the number of vessels. Data within each group were analyzed using ANOVA for nonparametric repeated measures, and, when appropriate, post hoc analyses performed with the Dunn's multiple-comparison tests. Microvascular pressure was analyzed using two-way ANOVA (diameter and fluid); post hoc analyses were performed with Bonferroni post tests. All statistics were calculated using GraphPad Prism 4.01 (GraphPad Software; San Diego, CA). Changes were considered statistically significant if P < 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
Eleven animals were entered into this study, and all animals tolerated the entire hemodilution protocol without visible signs of discomfort. The animals were assigned randomly to the experimental groups of hemodilution with {alpha}{alpha}Hb (n = 5) and hemodilution with MP4 (n = 6). (Availability of the {alpha}{alpha}Hb compound was limited and sufficient for only 5 experiments.) The results of the actual study were compared with control (no hemodilution) and those of a previous study (4) carried out under identical circumstances using PBH solution.

Data groups. One-way ANOVA on baseline, level 1, and level 2 systemic and microcirculatory data from the two groups showed no significant differences (minimum P = 0.06). Because the animals were found to be from the same population, this allows for grouping of the data into one representative group at each of the hemodilution levels before the use of the two test solutions. Therefore, in our study, baseline and level 2 (n = 11) were statistically compared with the experimental groups at level 3 exchange transfusion with {alpha}{alpha}Hb (n = 5) and MP4 (n = 6).

Systemic parameters. The two experimental groups treated with the exchange protocol showed a significant reduction in Hct after each exchange, 50.1 ± 2.0% for baseline, 27.5 ± 1.6% for level 1, 18.5 ± 1.6% for level 2, 11.1 ± 0.9% for level 3 MP4, and 11.2 ± 0.7% for level 3 {alpha}{alpha}Hb (P < 0.001 for all level 3 groups compared with baseline). {alpha}{alpha}Hb showed the same trend up to and including level 3 hemodilution, namely, 15.6 ± 1.1 g/dl for baseline, 9.2 ± 0.5 g/dl for level 1, and 6.1 ± 0.7 g/dl for level 2. For level 3 hemodilution, total {alpha}{alpha}Hb was 4.8 ± 0.4 g/dl for MP4 and 5.6 ± 0.4 g/dl for {alpha}{alpha}Hb (P < 0.001 for all level 3 groups compared with baseline). Information on exchange using PBH was reported previously (4). Level 3 exchange with PBH produced a blood hemoglobin content of 6.7 ± 0.7 g/dl, which was similar to the level 2 exchange (6.0 ± 0.8 g/dl) and was significantly higher than that with MP4 and {alpha}{alpha}Hb because of the higher hemoglobin concentration in the PBH solution.

MAP was not changed from baseline (99.6 ± 7.6 mmHg) after the level 1 exchange (92.5 ± 10.3 mmHg) and decreased upon further hemodilution with Dextran 70 to 87.2 ± 9.8 mmHg at level 2. MAP decreased to 82.4 ± 6.9 mmHg (P < 0.05 vs. baseline) after level 3 with MP4, whereas level 3 exchange with {alpha}{alpha}Hb did not show a significant change in MAP (94.4 ± 6.2 mmHg). HR was not affected significantly during the hemodilution protocol.

Changes in the systemic and blood gas parameters before hemodilution and after extreme hemodilution for experimental groups are presented in Table 2. Systemic arterial blood gas analysis showed a statistically significant rise in PaO2 from baseline after level 3 in all of the solutions (MP4, 86.2 ± 8.4 mmHg; {alpha}{alpha}Hb, 84.4 ± 9.6 mmHg, P < 0.05 vs. baseline). PaCO2, blood pH, and arterial base excess fell, but the number of animals was not high enough to establish statistical significance.


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  		Table 2. Macrohemodynamic parameters before and after blood exchange

 
Table 3 shows the plasma viscosity and COP attained at the end of the experiments. The similarity of COP suggests that their value is primarily determined by autotransfusion caused by the concentration of dextran in solution, which is calculated to be ~4.5% upon infusion. The difference in COP between MP4 and {alpha}{alpha}Hb may also be a factor when they are introduced in a 35% exchange. COP is determined by the total number of colloidal molecules occupying a given volume of solvent; thus neither MP4 nor {alpha}{alpha}Hb are diluted by about one-third in their oncotic capacity at the level 3 exchange transfusion, because they are diluted in a fluid in which there are already several active oncotic species, primarily Dextran 70 and albumin. Thus autotransfusion may be a more prominent factor in the MP4 exchange than in the {alpha}{alpha}Hb exchange due to the higher COP of MP4. In this, as in previous studies, there was no indication of dextran-hemoglobin-RBC interactions resulting in RBC aggregation.


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  		Table 3. Rheological properties and COP

 
Microhemodynamics. The changes in diameter, RBC velocity, and blood flow of arterioles (range 32–96 µm) and venules (range 33–96 µm) were measured after each hemodilution step. After level 3 exchange transfusion with MP4 and {alpha}{alpha}Hb, there was a slight arteriolar vasoconstriction to 0.98 ± 0.22 of baseline for MP4 (N = 20) and 0.96 ± 0.18 of baseline for {alpha}{alpha}Hb (N = 18). However, none of the changes were statistically significant. Arteriolar diameters measured after the administration of the solutions are presented in Fig. 1A normalized to baseline data.



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  		Fig. 1. Changes in arteriolar and venular hemodynamics after extreme hemodilution [hematocrit (Hct) 11%]. The dotted line represents the baseline level; N is the number of vessels studied. Arteriolar (A) and venular (B) diameters (means ± SD) in each animal group were as follows: level 1 (arterioles: 55.6 ± 16.2 µm, N = 36; venules: 66.2 ± 24.5 µm, N = 40); level 2 (arterioles: 58.5 ± 17.4 µm; venules: 70.6 ± 25.2 µm); level 3 surface-modified polyethylene glycol-conjugated human hemoglobin (MP4; arterioles: 56.2 ± 15.8 µm, N = 18; venules: 70.4 ± 20.4 µm, N = 20); level 3 {alpha}{alpha}-cross-linked human hemoglobin ({alpha}{alpha}Hb; arterioles: 56.5 ± 19.4 µm, N = 18; venules: 68.6 ± 26.1 µm, N = 20); and level 3 polymerize bovine hemoglobin (PBH; arterioles: 51.1 ± 14.6 µm, N = 18; venules: 58.5 ± 19.6 µm, N = 20). Arteriolar (C) and venular (D) red blood cell (RBC) velocities (means ± SD) in each animal group were as follows: level 1 (arterioles: 4.4 ± 2.5 mm/s, venules: 1.6 ± 0.9 mm/s); level 2 (arterioles: 4.6 ± 2.7 mm/s, venules: 1.6 ± 1.3 mm/s); level 3 MP4 (arterioles: 4.1 ± 1.8 mm/s, venules: 1.2 ± 0.8 mm/s); level 3 {alpha}{alpha}Hb (arterioles: 4.6 ± 2.5 mm/s, venules: 1.3 ± 1.1 mm/s); and level 3 PBH (arterioles: 3.4 ± 2.6 mm/s, venules: 1.2 ± 0.8 mm/s). {dagger}P < 0.05 relative to baseline. [PBH data are from the study of Cabrales et al. (4).]

 
Venular changes due to the hemodilution protocol are shown in Fig. 1B as a function of blood hemoglobin content. Level 3 exchange using MP4 and {alpha}{alpha}Hb dilated venules to 1.12 ± 0.15 (MP4, N = 20) and 1.04 ± 0.18 ({alpha}{alpha}Hb, N = 20) of baseline.

Figure 1, C and D, shows the change in RBC velocity in arterioles and venules as a function of blood hemoglobin content. A decrease in arteriolar RBC velocity was detected after level 3 exchange with MP4 and {alpha}{alpha}Hb, reducing arteriolar flow velocity to 0.84 ± 0.38 (MP4) and 0.75 ± 0.36 ({alpha}{alpha}Hb) of baseline, respectively. Venular RBC velocity increased after level 3 exchange to 1.05 ± 0.38 with MP4 and to 1.02 ± 0.37 with {alpha}{alpha}Hb of baseline, respectively.

Arteriolar and venular blood flow after the hemodilution protocols with the different test materials are presented in Fig. 2. Level 3 exchange with MP4 and {alpha}{alpha}Hb maintained arteriolar blood flow at baseline levels.



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  		Fig. 2. Arteriolar (left) and venular (right) blood flow after extreme hemodilution. Data are presented as means ± SE relative to baseline levels. The dotted line represents the baseline level. Flow in each animal group was as follows: level 1 (arterioles: 10.7 ± 2.8 nl/s, venules: 4.9 ± 1.8 nl/s); level 2 (arterioles: 12.1 ± 2.7 nl/s, venules: 5.7 ± 2.4 nl/s); level 3 MP4 (arterioles: 9.6 ± 2.9 nl/s, venules: 4.5 ± 1.5 nl/s); level 3 {alpha}{alpha}Hb (arterioles: 11.0 ± 3.1 nl/s, venules: 4.6 ± 1.8 nl/s); and level 3 PBH (arterioles: 7.1 ± 2.6 nl/s, venules: 3.1 ± 1.6 nl/s). {dagger}P < 0.05. [PBH data are from the study of Cabrales et al. (4).]

 
FCD. FCD (Fig. 3) was reduced after level 1 exchange in all groups to 92 ± 6% (P < 0.05 relative to baseline). Level 2 exchange reduced FCD to 85 ± 7% (P < 0.05 relative to baseline). FCD was further reduced after level 3 exchange for all test solutions (MP4, 70 ± 9%; {alpha}{alpha}Hb, 62 ± 8%; both P < 0.05 relative to baseline). There was no statistical difference in FCD between MP4 and {alpha}{alpha}Hb at level 3.



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  		Fig. 3. Functional capillary density (FCD) after extreme hemodilution with different hemoglobin-based oxygen carriers. ns, Not significant. [PBH data are from the study of Cabrales et al. (4).]

 
Microvascular pressure. Microvascular pressure distribution in the awake hamster window chamber preparation during control (nonhemodiluted) conditions in arterioles ranging 30–90 µm and small collecting venular vessels ranging 30–90 µm is shown in Fig. 4. All microvessels (arterioles and venules) were grouped according to their diameter, showing a significant decrease in pressure compared with control (P < 0.05 relative to control). The comparison of level 3 pressure distributions for the hemoglobin products shows a significant difference between MP4 and {alpha}{alpha}Hb microvascular pressure only in arterioles with a diameter of 80 µm (P < 0.05). The comparison of level 3 hemodilution with MP4 and the previous obtained data using PBH (4) shows that there is a significant higher pressure in arterioles with 80 µm diameter and lower over all the venules (P < 0.05).



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  		Fig. 4. Microvascular pressure distribution (means ± SD) after level 3 exchange hemodilution. All microvessels (arterioles and venules) were grouped according to diameter. Significant changes are presented (P < 0.05). [PBH data are from the study of Cabrales et al. (4).]

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 DISCLOSURES
 REFERENCES
 
The principal finding of this study is that capillary pressure is directly related to FCD independent of the mechanism by which capillary pressure changes (Fig. 5). According to a previous study (4), capillary pressure in extreme isovolemic hemodilution (Hct 11%) in the hamster window chamber model is reduced from the control range of 22–26 to 11–14 mmHg, when plasma and blood viscosities had values of 1.38 and 2.12 cp, respectively. Performing an identical level of reduction in Hct with {alpha}{alpha}Hb and MP4 in the present study (plasma and blood viscosity being about 1.35 and 1.80 cp, respectively) leads to a significantly increased capillary pressure and FCD. Conversely, the use of PBH in the same hemodilution protocol yields approximately the same respective plasma and blood viscosities; however, capillary pressure was in the range of 12–16 mmHg and FCD was reduced to 47 ± 12%.



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  		Fig. 5. Data for each animal on FCD and capillary pressure from this study compared with those of a previous study using Dextran 500, Dextran 70, and PBH after level 3 exchange. [PBH, Dextran 70, and Dextran 500 data are from the study of Cabrales et al. (4).]

 
The results obtained with these different hemoglobin-based OCPEs present differences compared with previous results found using Dextran 70 (essentially identical plasma and blood viscosities). The disparities are the consequence of the increase in oxygen-carrying capacity produced by the increased hemoglobin concentration in plasma and the respective vasoactivity of the modified hemoglobins. A Hct of 11% for the hamster appears to be slightly beyond the critical oxygen-carrying capacity limit; therefore, small increases in oxygen-carrying capacity should have notable effects, particularly in cardiac function, flow, and restoration of blood pressure. This effect was evident in comparing MAP attained with Dextran 70, which was 58 ± 9 mmHg, versus the values obtained with the hemoglobin solutions, which were in the range of 82–94 mmHg. Ideally, these experiments would have been carried out using identical concentrations of molecular hemoglobin; however, the disparity in colloidal oncotic properties between formulations would introduce a major variable in terms of blood volume responses if concentrations were equal. It should be noted that the primary clinical safety concern using these solutions is COP and volume regulation. Consequently, these solutions are designed to attain a clinically acceptable COP, which determine viscosity, whereas hemoglobin concentration and P50, PO2 tension at which hemoglobin is 50% saturated with oxygen, remain as variables.

The introduction of oxygen-carrying capacity via molecular hemoglobin in solution normalized systemic circulatory conditions with the microcirculatory effects that may be related to the vasoactivity of different hemoglobins. Vasoactivity in the present and previous study was only evidenced by the differences in microvascular measurements among {alpha}{alpha}Hb, MP4, and PBH, but not by the differences between {alpha}{alpha}Hb and MP4, with the exception of pressure in large arterioles (80 µm diamter), which was significantly lower than that found using {alpha}{alpha}Hb.

Capillary pressure was not measured directly because contact of the micropipette tip with 10- to 20-µm arterioles in this awake animal preparation during extreme hemodilution conditions causes a rapid contraction of the microvessel. This phenomenon was not seen during cannulation of venules and larger diameter arterioles. Thus our reported capillary pressures represent the average hydraulic pressure that straddles the entrance and exit of the capillary circulation and is indicative that in this tissue the net driving pressure across the capillary network is on the order of 4 mmHg.

The difference in pressure between the smallest arteriolar and the collecting venules was 6.9 ± 3.1 for MP4, which had an average capillary pressure of 18.6 ± 3.4 mmHg, and 6.5 ± 2.7 for {alpha}{alpha}Hb, which had an average capillary pressure of 16.1 ± 3.3 mmHg. In a previous study (4), we determined that the same protocol performed with the high-viscosity plasma expander Dextran 500 yielded results similar to those found for MP4. This similarity suggests that the effects found with MP4 may also be in part related to the physical properties of these molecules and particularly their respective dimension because they are both large molecules, as suggested by Sakai et al. (20), who found an inverse correlation between vasoactivity and molecular dimensions.

Sustained capillary pressure in this study indicates that MP4 and {alpha}{alpha}Hb are able to provide tissue perfusion through the maintenance of FCD, a phenomenon also documented by the measurement of FCD and flow in the study of Tsai et al. (28). It is notable that increasing or restoring blood pressure by means of a vasoactive material, such as PBH, does not result in either the restoration of capillary pressure or the restoration of FCD, whereas {alpha}{alpha}Hb appears to not have the same effect.

The difference in microvascular effects found between {alpha}{alpha}Hb and PBH may also be related to the difference in plasma concentration attained by these molecules, namely, 1.8 versus 3.6 g hemoglobin/dl blood, a factor that may also be responsible for the different responses in changes of peripheral vascular resistance in the arteriolar microcirculation. Data on pressure redistribution and blood flow allows us to compute the distribution of vascular resistance in the microcirculation, as shown in Fig. 6. This calculation shows that most of the vasoactivity affects arterioles greater than about 50 µm in diameter. It is also notable that the vasoactive response of PBH appears to be located in vessels of about 70 µm in diameter, whereas the location of vasoactivity for {alpha}{alpha}Hb may be small arteries, and not in the microcirculation. Figure 6 also shows that vascular resistance for MP4 is decreased relative to control, a phenomenon that is in part due to the lowered blood viscosity and also to the lack of localized changes in vascular resistance in the microcirculation.



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  		Fig. 6. Distribution of vascular resistance for each segment of the microvascular network. The peak vascular resistance found for PBH in 70-µm arterioles is in contrast with the lower vascular resistance found with {alpha}{alpha}Hb, suggesting that the vasoconstrictor effect of the later occurs in larger arterial vessels. The pattern of vascular resistance found for MP4 indicates the absence of a specific locality for vasoactivity. Furthermore, this parameter is lower relative to control due the decreased blood viscosity. {Delta}P, change in pressure. [PBH, Dextran 70, and Dextran 500 data are from the study of Cabrales et al. (4).]

 
In conclusion, this study supports the concept of FCD as being a linear function of capillary pressure, which is related to central blood pressure in addition to the tone of the microvasculature resistance altered by the vasoactivity of the hemoglobin formulation. Restitution of oxygen-carrying capacity with a molecular hemoglobin solution upon passing the transfusion trigger produces a condition of extreme hemodilution in terms of blood and plasma viscosity. Low viscosity and vasoactivity can result in pathologically low central and capillary pressures affecting FCD. The use of any of the modified hemoglobins tested is shown to be able to restore central blood pressure in conditions of extreme hemodilution. Capillary pressure was maintained to near normal levels when {alpha}{alpha}Hb and MP4 were used to restore oxygen-carrying capacity but was significantly decreased in extreme hemodilution with PBH, a result that was paralleled by the level of recovery of capillary pressure and FCD. These findings have implications in the design of blood substitutes because MP4 has the lowest hemoglobin concentration relative to the other two materials tested. Because both MP4 and the previously tested hemodiluent Dextran 500 in an identical protocol produced nearly identical results, it is possible that the effects of these molecules are in part due to their large dimensions. However, a previous study (28) has shown that MP4 delivers significantly more oxygen to the tissue as a consequence of its oxygen-carrying capacity and P50 characteristics. There was no difference in vasoactivity in terms of measurements of FCD between {alpha}{alpha}Hb and MP4; however, calculation of the segmental hydraulic vascular resistance across the microcirculation showed that this parameter was consistently below control for MP4 and above control for both {alpha}{alpha}Hb and PBH.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
DISCLOSURES
 REFERENCES
 
This study was supported by National Heart, Lung, and Blood Institute Bioengineering Research Partnership Grant R24-HL-64395, Grants R01-HL-62354 and R01-HL-62318 (to M. Intaglietta), and Grant R01-HL-64579 (to R. M. Winslow).


   DISCLOSURES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
REFERENCES
 
R. W. Winslow is the President, CEO, and Board Chairman of Sangart Incorporated, San Diego, CA.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. Cabrales, Dept. of Bioengineering, 0412, Univ. of California-San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0412 (E-mail: pcabrales{at}ucsd.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 

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