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1Department of Anesthesiology and Critical Care Medicine, The Johns Hopkins University, Baltimore, Maryland; and 2Department of Chemistry, Temple University, Philadelphia, Pennsylvania
Submitted 28 October 2005 ; accepted in final form 13 January 2006
ABSTRACT
Cell-free hemoglobin solutions with high oxygen affinity might be beneficial for selectively delivering oxygen to ischemic tissue. A recombinant hybrid hemoglobin molecule was designed using the human 
-subunit and the bovine 
-subunit, with placement of surface cysteines to permit disulfide bond polymerization of the tetramers. The resulting protein generated from an Escherichia coli expression system had a molecular mass >1 MDa, a P50 of 
3 Torr, and a cooperativity of n = 1.0. Anesthetized mice were transfused during 2-h occlusion of the middle cerebral artery. Compared with transfusion with 5% albumin, cerebral infarct volume was reduced by 41% with transfusion of a 3% solution of the high oxygen-affinity hemoglobin polymer and by 50% with transfusion of a 6% solution of the polymer. Transfusion of a 6% solution of a 500-kDa polymer possessing a P50 of 17 Torr and a cooperativity of n = 2.0 resulted in a 66% reduction of infarct volume. These results indicate that cell-free Hb polymers with P50 values much lower than that of red blood cell hemoglobin are highly capable of salvaging ischemic brain. The assumption that the P50 of blood substitutes should be similar to that of blood might not be warranted when used during ischemic conditions.
blood substitute; cerebral ischemia; middle cerebral artery; oxygen carrier
We previously reported the characterization of a polymer of human hemoglobin, Hb Prisca (6), and of a recombinant hybrid Hb with the -subunit of human HbA and the -subunit of bovine Hb (2). The latter has, in Escherichia coli, a higher expression level, which allowed us to produce this recombinant Hb in an amount sufficient for transfusion in mice. Appropriate amino acid substitutions were introduced to replace the naturally occurring cysteine residues while introducing a novel Cys residue at position 9 in the -chains on the protein surface. These substitutions resulted in the formation of intermolecular disulfide bonds that generate a nondissociable polymer that consists of 7–8 Hb tetramers (2). This polymer, designated Hb Polytaur, has a P50 of 17 Torr and a cooperativity of n = 2.0. Transfusion of Hb Polytaur in mice produced small increases in arterial blood pressure that were similar to those obtained with transfusion of albumin. However, when transfusion occurred after middle cerebral artery occlusion (MCAO), mice transfused with a 3% solution of Hb Polytaur had a 22% reduction in infarct volume (2). Thus Hb Polytaur appears to be capable of delivering O2 to ischemic tissue, despite a low P50 of 17 Torr. This result raised the possibility of whether Hb molecules possessing much lower P50 than that of red blood cell Hb can be an effective O2 carrier in ischemic tissue.
The purpose of the present study was to determine whether a recombinant Hb polymer, possessing functional characteristics similar to myoglobin with very low P50 and absent cooperativity, would be capable of reducing infarct volume from focal cerebral ischemia. High molecular weight polymers were obtained when the surface Cys were introduced without the substitution of the naturally occurring Cys. By retaining naturally occurring Cys residues, a large heterogeneous polymer, designated Hb (Polytaur)n, was derived. Because it has been suggested that high concentrations of plasma Hb might deliver too much O2 to precapillary arterioles and cause constriction, we tested the effect of two concentrations of Hb (Polytaur)n, 3 and 6%. In addition, the effect of transfusing a 6% solution of Hb Polytaur was compared with previous results with a 3% solution of Hb Polytaur (2).
METHODS
Recombinant hemoglobin.
The design of DNA plasmids used for expression of the human -subunit and bovine -subunit of the hybrid Hb in E. coli grown in a 5-liter fermentor as well as the steps for protein purification of the tetramers and polymerization of the tetramers have been described (2). All mutations were made using the pAlter system (Promega, Madison, WI). The -globin gene carrying the desired mutations was excised from the pAlter plasmid with NcoI and BssHII and inserted into pNFBovine (2) that had been digested with the same two enzymes. The -globin gene of pDLHBV (2) was replaced with the mutated -globin gene, using NcoI and HindIII to yield the plasmids expressing Hb Polytaur and Hb (Polytaur)n. To construct Hb Polytaur, a cysteine residue was substituted for the 9Ser. This substitution permitted spontaneous formation of stable disulfide bonds among the tetramers. In addition, the 104Cys was replaced by Ser, and the 93Cys was replaced by Ala to prevent spurious disulfide bond formation during protein refolding and to permit the formation of a relatively homogeneous polymer of 500 kDa. To obtain Hb (Polytaur)n, the 104Cys and 93Cys were left intact, resulting in polymers of heterogeneous sizes >1 MDa. The purified Hb proteins were dialyzed against lactated Ringer solution. The solution was filtered (0.45 µm), and endotoxin was removed using Detoxy-gel (Pierce, Rockford, IL).
Experimental protocol.
Because of the limited capacity of the fermentor to produce recombinant protein sufficient for physiological experiments, studies were performed on mice. All procedures were approved by the Johns Hopkins University Animal Care and Use Committee. Male C57Bl/6 mice were anesthetized with 1.5% halothane via face mask. Rectal temperature was maintained with a heating lamp. A femoral artery was catheterized. Focal cerebral ischemia was produced by advancing a single monofilament through the internal carotid artery to the origin of the middle cerebral artery (7). The tip of a 6-O monofilament was blunted by heat and coated with cyanoacrylic glue. An incision was made in the neck for isolating the right carotid artery. The filament was inserted into the internal carotid artery through a cut stump of the external carotid artery, and the tip was advanced 6 mm past the junction of the pterygopalantine artery to produce MCAO. Starting at 10 min after MCAO, a hypervolumetric exchange transfusion was performed to reduce hematocrit from 44 to 36% over a 35-min period by infusing 0.95 ml in four-step increments, while alternating with the withdrawal of 0.7 ml in three-step increments from the femoral artery. In preliminary experiments, the increase in mean arterial pressure with transfusion of the Hb (Polytaur)n solution (17 ± 3 mmHg) was similar to that with Hb Polytaur (16 ± 9 mmHg) and albumin (11 ± 5 mmHg) solutions. After 45 min of MCAO, the catheter was removed, incisions were closed, and halothane administration was discontinued. Neurologic deficit was evaluated, and mice without circling behavior were excluded. At 2 h of MCAO, the mouse was briefly anesthetized, and the monofilament was withdrawn from the internal carotid artery to permit reperfusion. At 24 h, the brain was harvested and cut into 2-mm coronal slabs for vital dye staining with a 1% solution of 2,3,5-triphenyltetrazolium chloride. The volume of the infarct was integrated over five slabs and corrected for swelling (7).
In one experiment, mice were transfused with either a solution containing 5% albumin (n = 6), 3% Hb (Polytaur)n (n = 7), or 6% Hb (Polytaur)n (n = 5). In a second experiment, mice were transfused with either a solution containing 5% albumin (n = 7), 3% Hb Polytaur (n = 7), or 6% Hb Polytaur (n = 5). For each polymer, infarct volume was compared among the three groups that were transfused with albumin and the 3 and 6% Hb solutions by analysis of variance and the Newman-Keuls multiple range procedure, with significance set at the P < 0.05 level. Data are presented as means ± SD.
RESULTS
Recombinant Hb (Polytaur)n possessed a P50 of 3 mmHg and a Hill coefficient of n = 1 (Fig. 1). Exchange transfusion with a 3% solution of Hb (Polytaur)n during MCAO markedly reduced infarct volume to 59% of that in the albumin-transfused group (Fig. 2). When the concentration of Hb (Polytaur)n was increased to 6%, infarct size was reduced to 50% of that in the albumin-transfused group. Infarct volume as a percent of the volume of the cerebral hemisphere was 58 ± 12% in the albumin-transfused group, 35 ± 17% in the group transfused with 3% Hb (Polytaur)n, and 28 ± 6% in the group transfused with 6% Hb (Polytaur)n. The values in the Hb-treated groups were significantly different from the value in the albumin-treated group whether absolute infarct volume or percentage of hemisphere values was analyzed. The value in the albumin-transfused group was not different from that in a group without transfusion (58 ± 5%).
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DISCUSSION
The major finding of this study is that exchange transfusion of cell-free recombinant Hb polymers with high O2 affinity during focal cerebral ischemia is capable of reducing infarct size. It has generally been assumed that cell-free Hb should have an O2 affinity similar to red blood cell-based Hb to provide optimal O2 delivery. However, the reduction in infarct volume with high-affinity Hb polymers implies that these polymers are capable of delivering O2 to ischemic tissue. In models of severe anemia and hemorrhagic shock, transfusion of high O2 affinity Hb was observed to produce better capillary perfusion and improved oxygenation in the hamster skin-fold microcirculation (16, 19), due in part to less arteriolar constriction and less precapillary O2 loss. A similar mechanism might be operative in metabolically active brain tissue. High O2 affinity might limit O2 loss across pial arterioles and conserve O2 delivery to tissue with low PO2.
Experimental studies of MCAO in rat demonstrated a beneficial effect of transfusion of a fumaryl cross-linked tetrameric Hb possessing a P50 of 32 Torr (1, 4, 5). However, this product was not efficacious in a clinical trial of stroke (13). Transfusion during MCAO of a sebacyl cross-linked tetrameric Hb possessing a P50 of 34 Torr failed to reduce acute injury volume in cats, possibly because improved intraischemic blood flow at reduced hematocrit was delayed by over 2 h after the transfusion (11). Polymerization of Hb tetramers reduces peripheral extravasation and the associated vasoconstriction (8). However, in many cases, polymerization can decrease cooperativity and P50, thereby resulting in an O2 carrier with properties analogous to myoglobin. With large-scale polymerization resulting in heterogeneous polymers >1 MDa, a P50 of 3 Torr and a cooperativity n = 1.0, Hb (Polytaur)n decreased infarct volume by 41% when transfused at a 3% concentration. This reduction was significantly greater than the 22% reduction seen with the 3% solution of Hb Polytaur possessing a P50 of 17 Torr and a cooperativity of n = 2.0. Thus transfusing a relatively dilute solution of a cell-free Hb with very low P50 unexpectedly produced a large reduction in infarct volume.
At a 6% concentration, both polymers reduced infarct size similarly by 50–66%. The large magnitude of this reduction with 2 h of MCAO suggests that transfusion of these O2 carriers during the first 45 min of MCAO maintains a viable state in a significant amount of tissue that would normally be in the core and penumbra regions of ischemia. The ability to salvage tissue with more delayed treatment was not currently examined. Because tissue mitochondrial PO2 is presumed to drop to critically low levels during cerebral ischemia, elevating O2 delivery by even modest amounts could have profound effects in ischemic regions with marginal levels of perfusion. Although regional blood flow was not measured autoradiographically in the present study, cell-free Hb may improve oxygenation independent of any increased perfusion that might occur at reduced hematocrit and blood viscosity. By increasing the effective solubility of O2 in the plasma, cell-free Hb can facilitate the diffusion of O2 from the red blood cell to the endothelium (9, 10). Furthermore, because of the corpuscular nature of red blood cells in a single line with interspaced plasma in capillaries, the addition of an O2 carrier in the plasma increases the effective surface area for O2 diffusion across the endothelium (17). In addition, the low perfusion pressure across the microcirculation during ischemia is thought to increase the heterogeneity of red blood cell flux among capillaries, although plasma flow is maintained in most capillaries (15, 18). Capillaries perfused with plasma and low red blood cell flux would have disproportionately improved O2 flux after cell-free Hb transfusion and more homogeneous O2 delivery. Thus cell-free Hb with low P50 theoretically can improve oxygenation in ischemic tissue by multiple mechanisms.
Increasing the concentration of the infused Hb from 3 to 6% salvaged additional tissue in the case of Hb Polytaur. However, the additional salvage with a 6% solution of Hb (Polytaur)n was not significantly greater than the 3% solution, presumably because the 3% solution was already salvaging most of the tissue outside the ischemic core, where perfusion is too low to benefit from an increase in O2 carrying capacity. Increasing the concentration of the fumaryl cross-linked tetrameric Hb to 20% was noted to produce an additional benefit (4). Higher concentrations were not tested in the present study because the production yield is reduced and our fermentor system has limited capacity for producing large quantities of the protein. Higher concentrations could be beneficial by 1) increasing the O2 delivery during MCAO, 2) extending the duration of elevated plasma of these Hbs, which have a circulating half-life of 3 h in rodents (2, 3), and 3) by increasing the oncotic pressure. The concern that a high concentration of transfused Hb might cause unwanted vasoconstriction and limit O2 delivery was not borne out by the present data on infarct volume in ischemic brain.
Therefore, the present data indicate that cell-free Hb polymers with P50 values much lower than that of red blood cell Hb are highly capable of salvaging ischemic brain. The assumption that P50 of blood substitutes should be similar to that of blood might not be warranted when used during ischemic conditions.
GRANTS
This work was supported by a grant from the National Institute of Neurological Disorders and Stroke (NS-38684) and by the Eugene and Mary B. Meyer Center for Advanced Transfusion Practice and Blood Research at the Johns Hopkins University School of Medicine.
ACKNOWLEDGMENTS
The authors are grateful to Sarah Texel for technical assistance and to Tzipora Sofare for editorial assistance.
FOOTNOTES
Address for reprint requests and other correspondence: R. C. Koehler, Dept. of Anesthesiology and Critical Care Medicine, The Johns Hopkins Medical Institutions, 600 North Wolfe St., Blalock 1404, Baltimore, MD 21287 (e-mail: rkoehler{at}jhmi.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
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