Hemoglobin (Hb)- and perfluorocarbon-based, oxygen (O2)-carrying solutions have been developed with the goals of improving O2 delivery, tissue oxygenation (tPO2), and patient survival (1). Hemoglobin-based O2 carriers (HBOCs) are considered to be superior to perfluorocarbons because they carry more O2 and do not require elevated O2 tension to increase O2 delivery (1). Most HBOCs possess physical characteristics (molecular structure, Hb concentration, P50, viscosity, oncotic pressure) similar to blood (2). The administration of a purified, polymerized, bovine Hb solution (HBOC-201), for example, improves tPO2 in skeletal muscle, liver, and central organs following profound isovolemic hemodilution; improves tPO2 in the brain after traumatic brain injury and hemorrhagic shock; reverses anaerobic metabolism; and improves survival in rats, hamsters, dogs, and pigs following experimental anemia, hemorrhage, or trauma (3-13).
Questions remain, however, concerning the mechanism and result of vasocontrictive effects of HBOCs, the development of systemic and pulmonary hypertension, and the potential for oxidative stress in human patients, thereby limiting potential benefits afforded by increases in blood O2-carrying capacity (14-17). Hemoglobin-based oxygen carrier-induced vasoconstriction has been attributed to nitric oxide scavenging and distortion of the nitric oxide diffusion field by plasma and extravascular molecular Hb and an oversupply of arteriolar O2 (18, 19). Furthermore, in vitro and in vivo studies suggest that the molecular size and O2 affinity of most Hb solutions in conjunction with their relatively low viscosity may predispose to autoregulatory vasoconstriction (20-23). Reports of arteriolar vasoconstriction and reduced cardiac output have led to the consensus opinion that conventional HBOCs may predispose to spatial heterogeneity in microcirculatory blood flow and a decrease in functional capillary density and tPO2 regardless of increases in the O2-carrying capacity of blood (16, 24, 25). Others, however, suggest that optimization of a low O2-affinity acellular Hb concentration (Oxyglobin; Biopure, Cambridge, Mass) increases tissue oxygenation without substantial vasoconstriction (13, 26). Indices of tissue oxygenation and tPO2 are currently considered the most specific and reliable quantitative markers of tissue perfusion (27). The objective of this study was to determine the effects of HBOC-201 on tPO2 in heart, brain, and kidney after extreme isovolemic hemodilution in a large animal model, anesthetized pigs. We hypothesized that extreme isovolemic hemodilution with HBOC-201 would not compromise tPO2 in vital organs.
MATERIALS AND METHODS
The study was approved and conducted in accordance with the guidelines established by the Institutional Laboratory Animal Care and Use Committee of The Ohio State University. A veterinarian was available 24 h·d−1, 7 d·wk−1 to provide care if required.
The study included forty 12- to 15-kg pigs purchased from Genetiporc USA (Alexandria, Minn). The pigs were identified by ear notch; group housed in climate-controlled, indoor pens; and offered Mazuri mini pig active feed. All pigs underwent a 7-day acclimation period before beginning the study. Prestudy laboratory evaluation included a physical examination, blood chemistry profile, and complete blood count to verify the health of the pigs. Food, but not water, was withheld after midnight on the morning of the procedure.
The pigs were randomly assigned into one of three groups. Group 1 (control) included four pigs that served as time controls and were anesthetized but not subjected to test procedures. The remaining 36 pigs underwent a 1:1 stepped isovolemic exchange transfusion representing 10%, 30%, and 50% of their total blood volume (70 mL·kg−1). Eighteen pigs (group 2, human serum albumin [HAS], n = 6/tissue for heart, brain, and kidney tPO2) were administered oncotically matched HSA (6 g·dL−1; Baxter Healthcare Corporation, Westlake Village, Calif), and 18 pigs (group 3, HBOC, n = 6/tissue for heart, brain, and kidney tPO2) were administered HBOC-201 (Biopure Corporation, Cambridge, Mass): mean molecular weight 250 kd, Hb 13 g·dL−1, and P50 40 mmHg. Blood was removed from a femoral arterial catheter followed by infusion of HSA or HBOC-201 through a jugular vein catheter. The initial volume exchange was 8 mL·kg−1 (10% of estimated blood volume) followed by two 15-mL·kg−1 (second exchange, 30%; then third exchange, 50%) exchanges (Fig. 1). The withdrawal rate was 1 mL·kg−1·min−1, and infusion rate was 0.5 mL·kg−1·min−1.
Each pig was immobilized with ketamine i.m. (15-20 mg·kg−1) and atropine i.m. (0.1 mg·kg−1) at least 3 h before beginning the study. Lactated Ringer's solution (2-4 mL·kg−1·h−1) was administered via an ear vein. General anesthesia was produced and maintained by administering sodium pentobarbital, 15 mg·kg−1 i.v. followed by 3- to 10-mg·kg−1·h−1. All pigs were oral-tracheally intubated, placed in a supine position, and ventilated with room air (21% O2) to maintain PaCO2 between 35 and 45 mmHg and PaO2 greater than 80 mmHg (ABL 725; Radiometer America, Westlake, Ohio). A dual-tipped pressure sensing catheter (Millar Instruments, Houston, Tex) was inserted into the surgically exposed left carotid artery and advanced into the left ventricle for measurement of left ventricular end-diastolic pressure (LVEDP; distal tip), and proximal aortic blood pressure (proximal tip). A polyethylene-160 catheter was inserted into the surgically exposed right femoral artery and advanced to the level of the diaphragm and used for blood withdrawal during exchange transfusion. A polyethylene-160 catheter was inserted into the surgically exposed left jugular vein and advanced into the right atrium. The left carotid artery and jugular venous catheters were used to obtain blood samples and administer test article, respectively.
Lithium naphthalocyanine (LiPc) microcrystals (O2 probe) were deposited at a 2- to 3-mm depth from the tissue surface by inserting a 25-gauge needle into the kidney or brain. The LiPc microcrystals produced a single sharp electron paramagnetic resonance (EPR) line, the width of which is highly sensitive and specific to the tissue partial pressure of O2 (28). The O2 sensitivity of the probe in saline was calibrated at L-band (1.3 GHz) by equilibrating with known partial pressures of O2. The calibration measurements demonstrated that the EPR line-width (ΔBpp) of the LiPc probe showed a linear response in the range of 0 to 150 mmHg. A slope of 0.89 μT·mmHg−1 was obtained for O2 sensitivity. The calibration data were used to convert the experimentally measured EPR line-widths into PO2 values (28). The area of tissue being measured was approximately 3 mm2.
Left ventricular myocardial tPO2 was determined by advancing a 100-μm diameter fiberoptic probe (Oximetric; Abbott, Munich, Germany) through an 18-gauge Teflon over-the-needle catheter after prepositioning of the needle in a relatively avascular (no visible vessels) area approximately 2 to 3 mm below the epicardial surface of the left ventricle. The fiberoptic probe tip was approximately 1 cm caudal to the left anterior descending (left interventricular) coronary artery and 2 cm ventral to the left circumflex coronary artery. Tissue tPO2 determinations were representative of local O2 tension at the probe tip. Proper positioning of the fiberoptic O2 sensing probe was determined by the lack of hemorrhage or discoloration at the site of probe placement, appropriate fluctuations in myocardial blood flow, and stable tPO2 and temperature values. Only probe insertions that demonstrated no periprobe hemorrhage and that produced stable tPO2 values were accepted for data analysis. Local tissue temperature was measured by a fully integrated thermocouple, allowing simultaneous monitoring of tissue partial pressure of oxygen (PtO2), temperature, and automatic temperature correction.
Data were obtained continuously and recorded 5 min before each exchange, 20 min after each exchange, and again at 2 h after the third (50%) exchange transfusion. One hour elapsed between exchange transfusions. Each data set included arterial and mixed venous hematocrit (Hct; %), total protein (TP; g·dL−1), red blood cell (RBC) and plasma Hb concentration (g·dL−1), methemoglobin concentration (metHb; %), pH and blood gases (PO2 and PCO2; mmHg; ABL Radiometer, Copenhagen, Denmark), O2 saturation (%), base excess (mmol·L−1), lactate (mmol·L−1), heart rate (beats·min−1), systolic arterial blood pressure, diastolic arterial blood pressure, mean arterial blood pressure (MAP; mmHg), LVEDP (mmHg), and body temperature (°C). Tissue O2 tension (tPO2; mmHg) was determined by EPR oximetry in kidney and brain and by fluorescence fiberoptic microprobe in the heart (8, 15). Initial measurements were performed for tPO2 immediately after stabilization of the oximetry probes and continuously throughout the experiments. All pigs were killed at the end of the experiment by administering sodium pentobarbital (150 mg·kg−1, i.v.) in the auricular vein.
Tissues were harvested from the heart, brain, and kidney. The ventricular myocardium, cerebrum, and kidney cortex were stained with hematoxylin-eosin to detail cellular structures. Trichrome stain was applied to distinguish nuclei (black), collagen (blue), and muscle (red) and von Kossa to determine areas of increased accumulation of calcium. The brain (cortex) was stained with Van Gieson to identify collagen. The tissues were prepared using standard techniques, and each specimen was microscopically evaluated (magnifications ×100-×400) using an objective mounted micrometer.
Results are expressed as mean (SD) for all groups. With the exception of tPO2, all hemodynamic, pH, and blood gas (PO2 and PCO2) parameters from each group (heart, brain, and kidney) were combined to provide a single comparison between HBOC versus HSA (Table 1). An ANOVA for repeated measures with Bonferroni correction for multiple comparisons was used to analyze all data with group and time serving as cofactors. Paired t tests were used to calculate differences from baseline within groups for all parameters other than tPO2. One-way ANOVA was used to determine differences among groups across time, followed by t tests. Statistical significance was assumed at P < 0.05. The statistical program used was Systat version 10.2 (Chicago, Ill).
Baseline hematology and blood chemical data were within normal limits for pigs. No differences in baseline values for any variable for any group were identified (data not shown). Hemodynamic and hematologic values did not change throughout the duration of the experiment in control-group pigs (Tables 1 and 2).
Heart rate increased and arterial blood pressure and LVEDP decreased during blood withdrawal. This pattern was reversed during infusion of either 6% HSA or HBOC-201. The transient hemodynamic changes associated with blood withdrawal and test article infusion are not reflected in Table 1 because hemodynamic data were obtained after the end of each infusion. Infusions of HBOC-201 produced sustained increases in MAP (23% Inf 1; 29% Inf 2; 35% Inf 3) from baseline values (Table 1). Mean arterial blood pressure did not change from baseline values after the administration of HSA. Arterial blood pressure was significantly greater in pigs administered HBOC-201 compared with HSA after the second and third exchange tranfusions and remained elevated for the duration of the experiment (Table 1).
Results of selected hematologic variables before and after transfusion with 6% HSA or HBOC-201 are presented in Table 2. Baseline values for Hct, TP, total blood Hb, RBC Hb, plasma Hb, and metHb concentrations were not different among groups and did not change from baseline in control-group pigs. The Hct decreased after the infusion of 6% HSA or HBOC-201. The TP did not change after the administration of 6% HSA and increased significantly to values ranging from 6 to 8 g·dL−1 in pigs administered HBOC-201 (Table 2). Total Hb values did not change throughout the experiment for pigs administered HBOC-201 but decreased from baseline values for pigs administered 6% HSA. The RBC Hb concentration decreased for all pigs administered 6% HSA or HBOC-201, whereas plasma Hb concentration did not change for pigs administered 6% HSA and increased after each of the three infusions of HBOC-201. Whole-blood metHb did not change in control pigs, and metHb remained less than 1% of total Hb in pigs administered 6% HSA and significantly increased to 3.6% (SD, 1.1%), with individual peak values ranging from 2% to 6% in pigs administered HBOC-201.
Changes in brain and kidney tPO2 remained relatively stable for the duration of the experiment after exchange transfusion with either 6% HSA or HBOC-201 (Table 3). The average tPO2 was significantly increased compared with baseline values in heart tissue after the first exchange transfusion with HBOC-201 (Table 3).
No significant histopathologic changes were observed in the brain, kidney, or heart tissue obtained from control pigs or in brain and kidney tissue obtained from pigs administered 6% HSA or HBOC-201. One and two small foci of coagulation necrosis, respectively, were observed in two of the six HBOC-201-treated pigs.
Exchange transfusion and hemodilution with 6% HSA or HBOC-201 to Hct values averaging 16% did not change or decrease tPO2 in the heart, brain, and kidney in a large animal model. Exchange transfusion produced predictable hematologic and hemodynamic effects based on the physical characteristics and component chemistry of the solution administered. Exchange transfusion with 6% HSA produced minimal or no changes in hemodynamics, pH, and blood gas variables other than those induced by hemodilution. The administration of HBOC-201 increased arterial blood pressure and plasma Hb concentration, maintained total blood Hb concentration, and reduced blood O2 saturation. Decreases in the Hct and RBC Hb concentration were attributed to the dilutional effects of 6% HSA or HBOC-201. Increases in TP after HBOC-201 administration were attributed to the concentration of protein infused (13 g·dL−1 Hb). The 2% to 6% increases in the percent whole-blood methemoglobin (ferric heme iron) were expected based on the known tendency for extracellular Hb to undergo spontaneous autoxidation (16, 29). The greater P50 and lower cooperativity of HBOC-201 compared with porcine RBC Hb were believed responsible for the observed decreases in the arterial and venous O2 saturation in pigs administered HBOC-201. None of these changes were of significant magnitude to jeopardize tPO2.
The average absolute increases in arterial blood pressure compared with baseline values after the administration of HBOC-201 were approximately 23%, 29%, and 35%. These values were greater than that produced by 6% HSA after each exchange transfusion, but are not considered to be abnormally elevated values for anesthetized pigs (12, 30). The largest change in arterial blood pressure (23%) occurred after the first exchange transfusion of HBOC-201, whereas subsequent infusions (30% and 50% of blood volume) did not produce proportional increases in arterial blood pressure. Importantly, we did not observe a significant decrease in any hemodynamic variable compared with control pigs. We adapted an anesthetic protocol known to preserve homeostatic responses in swine and produced concordant results to that study (30). Various anesthetic protocols, especially inhalant anesthetics, may be responsible for the conflicting results published by others because nearly all anesthetic drugs have the potential to produce smooth muscle relaxation and vasodilation and blunt the sympathoadrenal homeostatic responses to manipulations (31).
The small changes in macrohemodynamics observed in this study would not be expected to produce significant changes in tPO2 unless tissue blood flow, arterial blood O2 content (CaO2), or tissue metabolism was markedly altered at the microvascular (<30 μm) level. Decreases in tPO2 below the threshold for anaerobic metabolism (tPO2 <3 mmHg) would be expected to lead to anaerobic metabolism and increases in serum lactate, thus leading to the development of metabolic acidosis (32). Acid-base data and, in particular, the arterial lactate values we recorded suggest that tissue blood flow was adequately maintained by both treatment groups. Transient decreases and increases in tPO2 values were observed in all three vital organs during blood withdrawal and test article infusion, respectively, suggesting associated changes in tissue perfusion pressure. The tPO2, however, never fell below a critical value despite significant decreases in Hct after each exchange and was never below baseline values in any organ. The in vivo measurements of tPO2 in the heart, brain, and kidney support this inference. Increases in arterial blood pressure would be expected to maintain and potentially increase tPO2 because tissue perfusion pressure is a prime determinant of tissue blood flow, provided that microvascular resistance to blood flow does not increase significantly (24, 33). Our macrohemodynamic data (heart rate, arterial blood pressure, LVEDP) suggest that tissue perfusion pressure and blood flow were maintained, whereas the tPO2 data confirm the maintenance of tissue oxygenation in the presence of HBOC-201.
Our data conflict with previous studies that measured tPO2 in the skin fold of the cheek pouch of hamsters undergoing exchange transfusions using a related polymerized bovine-derived HBOC (HBOC-301) solution (24, 25). Although the previous and current studies used exchange transfusions to produce a similar degree of hemodilution, the earlier studies reported that exchange transfusion with HBOC-301 resulted in abnormally low tPO2 values and suggested that this effect may be attributable to nitric oxide scavenging, vasoconstriction, and hypoperfusion and could affect survival (25). There are several critical differences among these experiments and the current study. We measured tPO2 in three vital organs: heart, brain, and kidney, whereas the earlier studies measured tPO2 in a nonvital fatty, subcutaneous tissue or skeletal muscle of the hamster cheek pouch. Significant differences are likely to exist between the regulation of blood flow and tissue oxygenation between subcutaneous tissues and vital organs. It is likely that exchange transfusion-induced changes in the microcirculation of the hamster check pouch are indicative of shunting of blood away from nonvital organs, thereby decreasing tissue oxygenation. Our study indicates that the distribution and maintenance of blood flow and oxygenation to vital organs are maintained. We did not measure cardiac output, but based on the results of a previous study that used an anesthetic and experimental protocol almost identical to the current study, we believe that HSA (6 g·dL−1) and HBOC-201 markedly increased or maintained cardiac output, respectively (30). That study attributed increases in cardiac output and vital organ blood flow (heart, brain, kidney), produced by the infusion of HSA, to a compensatory response to hemodilution and decreased O2 content to maintain O2 delivery (30). The authors concluded that physiologic coupling of O2 delivery and consumption is maintained after administration of HBOC-201 and not impaired by local vasoconstriction (30). Furthermore, criticisms have been raised concerning the methodological limitations of the quenching method used in these earlier studies to determine tPO2 (34). Finally, and of greater importance, more recent data have demonstrated that the administration of a dilute solution of bovine-derived HBOC-301 (4 g·dL−1) restores tissue perfusion and functional capillary density and significantly improved tPO2 and acid-base parameters in the hamster check pouch window chamber model subjected to moderate hemorrhagic shock (50% bleed) (13, 26). This observation is consistent with our data in anesthetized swine and experiments, which examined the effects of HBOC-201 as salvage therapy for severe neurotrauma and polytrauma (12). The administration of HBOC-201 in this latter study reduced pressor and fluid requirements, improved intracranial pressure, and increased brain tissue tPO2 (12).
Our study clearly demonstrated that HBOC-201 is capable of maintaining tissue oxygenation in a clinically relevant large animal model in which the Hct was reduced by approximately 50% due to hemodilution. We chose the pig because of its cardiovascular and physiologic similarity to humans. The model was not designed to test the limits of the O2-carrying ability of HBOC-201, and a more severe model using alternative anesthetic drugs or anesthetic techniques may have modified our results. Our experimental model was developed to obtain a total volume exchange that mimicked previously published articles wherein hemorrhage or isovolemic hemodilution was carried out to remove 50% of the animals total blood volume or attain a cumulative 50% volume exchange (4-6, 9-11). These earlier studies suggested that this "stress" would result in the development of a metabolic acidosis (lactic acidosis) presumably from inadequate O2 delivery or maldistribution of blood flow and low tissue O2 tension. A recent study, using an experimental design similar to the current study, however, suggested that physiologic coupling of O2 delivery and consumption after administration of HBOC-201 is not impaired (30). Our study extends this conclusion and suggests that tPO2 is maintained, at least in the tissues (brain, kidney, heart) that we interrogated. Additional studies will need to be conducted to more definitively examine tissue blood flow and oxygenation in animals subjected to a more severe degree of isovolemic hemodilution (Hct = 10%, 5%). There may be a spectrum of critical Hct below which various transfusion fluids are unable to maintain tissue oxygenation. The tPO2 values recorded in the current study are similar to tPO2 values reported in other EPR studies and those using polarographic electrodes and fluorescence probes (6, 28). The EPR method is highly specific and sensitive but is similar to other technologies in that it is limited to small superficial areas of tissue and is difficult to use in vivo. Interestingly, the heart tPO2 remained more stable in pigs administered HBOC-201 compared with pigs administered 6% HSA. Potential explanations for this difference include the greater total O2-carrying capacity and content of blood containing HBOC-201 and differences in microcirculatory blood flow. The PtO2 values for brain and kidney were lower than those for heart in both the HSA and HBOC groups but similar to values reported for gut and skeletal muscle (3-6). The reason for these differences cannot be determined from our studies but may be related to differences in the techniques used to measure PtO2 and differences in the factors that regulate individual organ blood flow and blood flow distribution or differences in tissue metabolism. Finally, a review of the effects of HBOC-201 administration on organ (myocardium, lungs, hepatic parenchyma, jejunum, renal cortex/medulla) injury after moderate (40%) and severe (55%) controlled hemorrhage or severe uncontrolled hemorrhage in swine concluded that there is limited evidence for HBOC-201-induced oxidative stress and that observed histological changes are clinically equivocal (35). Our exchange transfusion experiment replaced 50% of the pigs' blood volume with HBOC-201 and was not preceded by hemorrhagic shock; therefore, it is not surprising that we did not observe consistent histopathologic changes of oxidative stress. One study has demonstrated that intracoronary infusion of preoxygenated HBOC-201 preserved myocardial metabolism, regional O2 delivery, and left ventricular function produced by a 3-min coronary artery occlusion (36). Importantly, intracoronary infusion of nonoxygenated HBOC-201, Ringer's solution, or no treatment resulted in immediate loss of ventricular function in the ischemic region and evidence for anaerobic metabolism (36). These findings have important clinical implications regarding the potential benefits of HBOC-201 as an organ-preservation fluid and require further investigation.
Taken together, our study supports and extends investigations that have identified that systemic alterations produced by HBOC-201 do not alter regional blood flow and are unlikely to impair vital organ (heart, brain, and kidney) oxygenation. There was no indirect evidence of altered tissue perfusion (normal serum lactate concentrations) and minimal, if any, evidence for tissue histopathologic changes that could be directly attributed to oxidative stress.
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