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1Department of Orthopedic, Plastic and Hand Surgery, Inselspital University Hospital, 3010 Berne, Switzerland; and 2Advanced Research Institute for Science and Engineering, Waseda University, Tokyo 169-8555, Japan
Submitted 1 April 2003 ; accepted in final form 5 May 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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blood substitutes; artificial red blood cells; microhemodynamics; hypoxia; collateral circulation
Oxygenation and survival of ischemic myocardial (7, 19), cerebral (3, 18, 29), and peripheral tissues (2) could successfully be improved after the infusion of solutions containing artificial oxygen carriers, such as perfluorocarbons and chemically modified Hbs. These solutions have initially been developed with the scope of reducing the need of allogeneic blood transfusions, and at least four compounds are currently in advanced clinical trials to evaluate their potential as red blood cell (RBC) substitutes in blood loss (1, 10).
In a recent study (6), we were able to demonstrate that hypoxia in ischemic, collateralized hamster flap tissue was attenuated by a 50% blood exchange with a solution containing Hb vesicles (HbVs) suspended in 6% Dextran 70 (Dx70). The effect was associated with an increased capacity to transport oxygen to the ischemic tissue, which was related to the presence of HbVs and to an improvement of microcirculatory blood flow.
The HbV consists of isolated, purified human Hb that is encapsulated with a double phospholipid membrane coated with polyethylene glycol (22). The encapsulation of Hb prolongs the circulation time in the organism and prevents direct contact of Hb with the endothelial lining, thus suppressing vasoconstriction due to NO scavenging, which has been attributed to chemically modified Hbs (20). Another major advantage of the HbV is that oxygen affinity may easily be adapted to the needs of the tissue by supplementing the appropriate amount of coencapsulated allosteric effector (pyridoxal 5'-phosphate) (24). It was shown that after a 80% blood exchange with HbV solutions, oxygen consumption of the microvasculature was near to normal for a P50 of 16 and 30 mmHg, respectively, whereas it was significantly reduced for a P50 of 9 mmHg. The development of artificial RBC substitutes has been characterized by the assumption that their oxygen affinity should be similar or lower than that of blood to facilitate the delivery of oxygen to the tissues in need. On the other hand, it has been postulated that shifting the oxygen dissociation curve of RBC-bound Hb to the left (26, 27) or applying artificial oxygen carriers with high oxygen affinity (11) may be beneficial for the oxygenation of hypoxic tissues, in which O2 diffusion from oxygen carrier to tissue is ensured by the high gradient of PO2.
The aim of this study was to test the effect obtained by increasing the oxygen affinity of HbVs on the oxygenation of the ischemic hamster flap tissue during normovolemic hemodilution with HbVs suspended in 6% Dx70. To this end, HbV with a P50 of 15 mmHg (HbV15) was compared with an HbV (P50 = 30 mmHg; HbV30) with an oxygen affinity similar to that of hamster blood (P50 = 28 mmHg). Furthermore, we wanted to evaluate the influence of the degree of normovolemic blood replacement with the HbV solutions. We chose a protocol that included three steps of hemodilution up to a level of 50% blood exchange, beyond which a clinical use does not appear to be reasonable.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Animal and flap preparation. The experiments were performed in a hamster skin flap model as described previously (4–6). Anesthesia was induced by pentobarbital injected intraperitoneally (100 mg/kg body wt, Nembutal, Abbott Laboratories; Chicago, IL). The carotid artery and jugular vein were cannulated for blood pressure monitoring and for the blood exchange and laboratory analysis, respectively. Catheterization and flap dissection were performed with the aid of an operating microscope at x10 magnification (Wild; Heerbrugg, Switzerland). After the animal was shaved and the back skin of the animal was epilated, the vascular anatomy was identified by diaphanoscopy. An island flap measuring 30 x 20 mm was dissected free from the surrounding tissue. The animal was then placed in a lateral postition on a specially designed Plexiglas stage providing a platform for mounting the flap, which was positioned with the skin lying on the platform and kept at its original size by sutures. The panniculus carnosus was meticulously removed except for a single layer of muscle tissue left in place to protect the vascular network. The flap was merely perfused via one artery and vein, which bifurcate into two equal-sized branches within the flap, each of them supplying a separate vascular territory. One of the branches was transected after being secured with microsurgical ligatures. Therefore, one vascular territory was anatomically perfused by the intact branch, whereas the other was indirectly perfused through the collateral vasculature connecting the two vascular networks. The raw surface of the flap was finally covered with a polyvinyl film to isolate the tissue from the environment. During surgery, 4 mg papaverine hydrochloride (Sigma; St. Louis, MO) dissolved in 1 ml physiological saline solution was applied to the pedicle by a soaked cotton tip to prevent vascular spasm.
Laboratory analysis. Blood samples were collected in 40 µl heparin-washed microtubes for measurement of total Hb concentration and arterial blood gases (ABL 625, Radiometer; Copenhagen, Denmark). Hematocrit was determined by centrifugation.
Microhemodynamic measurements. Investigations were performed using
an intravital microscope (Axioplan 1, Zeiss; Jena, Germany). Microscopic
images were captured by a television camera (intensified charge-coupled device
camera, Kappa Messtechnik; Gleichen, Germany), recorded on video (50 Hz,
Panasonic; Osaka, Japan), and displayed on a television screen (Trinitron
PVM-1454QM, Sony; Tokyo, Japan). The preparation was observed visually with a
x40 objective resulting in a total optical magnification of x909
on the videomonitor. Microvascular diameter was measured by transillumination
with a green filter, which gave a well-defined image of the width of the
erythocyte column. For the assessment of centerline velocity, white blood
cells (WBCs) were stained in vivo with rhodamine 6G (2 µmol/kg body wt iv,
Sigma). Fluorescence was visualized with the aid of an excitation filter
(530–560 nm), a dichroic mirror (580 nm), and a barrier filter (580 nm)
and through epi-illumination by a mercury lamp (Atto Arc, Zeiss)
(13). Velocity was calculated
by measuring the distance covered by the WBC during one time frame (20 ms).
Microvascular blood flow (Q) was calculated by means of Eq. 1
 | (1) |
The microvessels were classified according to physiological and anatomic features into conduit arterioles (connections to each other), end arterioles, and small venules (4, 12). The vessels were chosen for examination according to their optical clarity.
Tissue oxygen tension. Tissue PO2 was assessed with Clark-type microprobes consisting of polarographic electrodes and an oxygen-sensitive microcell (Revoxode CC1, GMS; Kiel, Germany). According to the manufacturer, the Revoxode CC1 provides reproducible values for several consecutive days without the need of recalibration. The length of the cell was 1 mm, and the sampling area was within 1 mm of the cell. The probes were inserted into the subcutaneous tissue in the center of each vascular territory under visual control and microscopic magnification. Care was taken to place the probes in such a way that no arterioles or large venules lay within the sampling area.
HbV solutions. The HbVs were prepared as previously reported (22, 24). They consisted of isolated human Hb encapsulated in a phospholipid vesicle coated with polyethylene glycol. The size of the vesicles was 253 ± 63 nm. The oxygen affinity (P50) was regulated by adding the coencapsulated allosteric effector pyridoxal 5'-phosphate, and it was calculated from the O2 equilibrium curve measured with a Hemox Analyzer (TCS Medical Products) at 37°C (24). The HbVs were suspended in 6% Dx70 (B. Braun Medical; Emmenbrucke, Switzerland). The physical characteristics of the solutions are summarized in Table 1. Oncotic pressure and viscosity were measured with a colloid osmometer (model 4420, Wescor; Logan, UT) and a cone-plate viscometer (PVII+, Brookfield Engineering; Middleboro, MA), respectively (30).
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Protocol. The animals were kept under light anesthesia with a
continuous infusion of 50 mg/ml pentobarbital given at a rate of 
0.5
mg·min–1·kg body
wt–1 throughout the experiment. The depth of
anesthesia was regulated by tolerance of a noxious reflex due to pinching of
the hind paw but no nonaversive reflexes (palpebral, corneal, and jaw reflex).
A constant temperature in the animal and flap preparation was maintained by
means of a heating pad and by keeping the room temperature at 28°C.
Normovolemic hemodilution was achieved by simultanous replacement of withdrawn
blood over 10 min. Hemodilution was performed in three steps at an interval of
1 h, thus reaching levels of 15%, 30%, and 50% of blood exchange. Measurements
were taken at the end of each interval.
Inclusion criteria were a normal vascular anatomy, sufficient optical clarity of the preparation, and a mean arterial pressure above 60 mmHg.
The animals were euthanized with an overdose of pentobarbital at the end of the experiment.
Statistical analysis. The InStat version 3 program (Graph Pad Software, San Diego, CA) was utilized for statistical analysis. If the assumption of a normal distribution was appropriate, data were presented as means ± SD; otherwise, they represented median and 25th and 75th percentiles. For normally distributed data, the time-related differences between repeat measurements and the differences between the groups were assessed by the paired and unpaired ANOVA, respectively, whereas the nonparametric Friedman and Kruskal-Wallis tests were used for not normally distributed data analysis. All tests were followed by the Bonferroni post test. A value of P < 0.05 was taken to represent statistical significance.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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The systemic data are summarized in Table 2. Similar hematocrits were obtained in all hemodiluted animals. After the 50% blood exchange, hemodilution with Dx70 resulted in a mean total Hb concentration of 6.5 g/dl, whereas the addition of HbV to the diluents enhanced the total Hb concentration to 8.7 and 8.0 g/dl, respectively (P < 0.05). Arterial PO2 was gradually raised after each step of hemodilution, reaching 71 mmHg in the Dx70 group (P < 0.01 vs. baseline) and 53 and 52 mmHg for HbV15-Dx70 [not significant (NS)] and HbV30-Dx70 (P < 0.05), respectively (both P < 0.05 vs. Dx70). Furthermore, the blood exchange was followed by gradual reductions of arterial PCO2 (P < 0.01 for Dx70; NS for the HbV groups) and increases in pH (NS).
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At baseline, the microhemodynamic data were similar in all groups. The diameters and centerline velocities for conduit arterioles, end arterioles, and venules in each part of the flap are summarized in Table 3. Mean centerline velocities were significantly reduced in the ischemic vessels compared with the anatomically perfused vessels (P < 0.01).
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The behavior of the microvascular diameters in both parts of the flap are shown in Fig. 1. A slight vasoconstriction was observed in the conduit arterioles after hemodilution with Dx70 (93 ± 10% in the anatomically perfused tissue and 95 ± 7% in the ischemic tissue, both NS) and HbV15-Dx70 (93 ± 9% anatomically perfused, NS), whereas the diameters remained virtually stable in the other microvessels in all groups.
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Microvascular blood flow did not show any relevant changes in the anatomically perfused vessels (Fig. 2), whereas it was significantly increased in all vessels in the ischemic tissue due to hemodilution (P < 0.05 for Dx70 and HbV15-Dx70; P < 0.01 for HbV30-Dx70). The highest values were obtained with the 30% and 50% blood exchanges, reaching 144% (108–160%) for Dx70, 166% (152–192%) for HbV15-Dx70, and 141% (118–165%) for HbV30-Dx70.
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Mean PO2 ranged between 23.0 ± 4.3 and 26.7 ± 2.2 mmHg in the anatomically perfused tissue. Tissue PO2 was significantly reduced in the ischemic part, where the values were between 7.7 ± 3.2 and 10.9 ± 3.4 mmHg (P < 0.01). In the anatomically perfused tissue, oxygenation was virtually not influenced by hemodilution with or without HbVs (Fig. 3). In the ischemic tissue, a transient improvement was observed during hemodilution with Dx70. The maximum was obtained after the 30% exchange (121 ± 17% of baseline, P < 0.05). Each step of blood exchange caused an increase in ischemic tissue oxygenation if the diluents contained HbV. Tissue PO2 was enhanced up to 217 ± 67% and 164 ± 33% for HbV15-Dx70 and HbV30-Dx70, respectively (both P < 0.01 vs. baseline and other groups).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Normovolemic hemodilution with Dx70 transiently improved the oxygenation in the ischemic tissue, reaching a peak of 121% of baseline at the 30% blood exchange. This level of hemodilution is considered to provide the highest RBC flux at the capillary level (15), thus resulting in maximal oxygen transport capacity both systemically (28) and in locally ischemic tissue (25). Furthermore, arterial PO2 was increased at this level of hemodilution due to hyperventilation. As we (5) demonstrated in previous experiments in the same model, increased arterial PO2 values were transferred as far as the collateral arterioles, which is where the blood circulation enters the ischemic tissue. In the present study, however, the improvement of oxygenation in the ischemic tissue was by far higher if HbV was added to the Dx70 solution, although microvascular blood flow, Hb concentration, and arterial PO2 were similar or lower. Moreover, the oxygenation was enhanced despite a simultaneous decrease in total Hb concentration due to hemodilution with the HbV-Dx70 solutions. These results suggest that under the given conditions, the presence of HbVs increases the capacity of blood to deliver oxygen to the ischemic tissue and that the effect is related to the level of blood exchange with HbV solutions.
The effect may be achieved due to the small size of the HbVs, which might allow them to perfuse capillaries that are no longer accessible to RBCs due to intraluminal obstructions or reduced perfusion pressure that have to be assumed in the ischemic, collateralized tissue in the present model. Indeed, circulating HbVs could be observed in capillaries that were no longer considered functional because of the cessation of RBC flux (23). However, our previous experiments (6) showed that the improvement of oxygenation in the ischemic tissue obtained after hemodilution with HbV solutions was dependent on an increased RBC flux in this tissue, which indicates that mechanisms different from the passage of HbVs through vascular obstructions may be present.
On the basis of previous intravascular oxygen tension measurements, it was estimated that 40–50% of the systemic arterial oxygen content exited the upstream vasculature before reaching the collateralized, ischemic flap tissue (5, 24). It has been shown in both experimental (8, 24) and theoretical (31) studies that oxygen delivery may be delayed in favor of the downstream vasculature if oxygen carriers with increased oxygen affinity are infused. It is conceivable that this effect was responsible for the increased ischemic tissue oxygenation obtained with HbV15-Dx70 in our study. The high oxygen affinity of HbV15 did not seem to hamper the unloading of oxygen to this tissue, which is promoted by the high oxygen tension gradient and the increased residence time of circulating blood (11). Furthermore, it may be assumed that oxygen delivery is facilitated due to metabolic acidosis, thus causing a shift of the oxygen dissociation curve to the right.
The results obtained with HbV30-Dx70 suggest that prevention of oxygen loss in the upstream vasculature may have been accomplished without raising the oxygen affinity of HbV. It has been shown that the diffusion of oxygen through the plasma may substantially be influenced by adding oxygen carriers (14, 16, 17). According to the Stokes-Einstein equation, the diffusivity of oxygen is inversely proportional to the size of the plasma-bound oxygen carrier and the viscosity of the suspension. In mathematical models and in vitro experiments, facilitated oxygen diffusion was ascribed to small-sized Hbs (14, 16, 17, 21), whereas, because of their large size, this effect was abolished if HbVs were used (21). Although not measured in our study, there is sufficient evidence to assume a marked increase in viscosity of the plasma suspension obtained during hemodilution with the HbV-Dx70 solutions, because an increase in plasma viscosity from 1.2 to 1.4 cP has been observed in hamsters after severe hemodilution with Dx70 (30), which has a threefold lower viscosity than HbV-Dx70. Taken together, it may be assumed that hemodilution with the HbV-Dx70 solutions caused a retention of oxygen in the upstream vasculature, which was related to both the size of the HbV and the increasing composite viscosity of the plasma suspension consisting in original hamster blood plasma, Dx70 molecules, and HbV, and which resulted in a shift of oxygen delivery in favor of the collateralized, ischemic and hypoxic flap tissue.
Additional studies will be necessary to outline the influence of both HbV concentration and viscosity of the solutions, and the long-term benefit of the observed improvement in oxygenation would yet have to be confirmed by evaluating the functionality and survival of the jeopardized tissue. Moreover, our data may not be extrapolated to ischemic conditions in other, vital organs, such as the myocardium or cerebral tissue, in which the oxygen demand is substantially higher than in the present flap tissue.
In summary, we conclude that up to a 50% blood exchange, normovolemic hemodilution with HbV-Dx70 solutions led to a dose- and oxygen affinity-dependent improvement of oxygenation in the ischemic, hypoxic flap tissue, which was not related to the oxygen-carrying capacity of the circulating blood. Thus our study strongly emphasizes the potential function of HbVs as a therapeutic that may be used to improve the delivery of oxygen to ischemic and hypoxic tissues, which exceeds its role as a simple RBC substitute.
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DISCLOSURES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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FOOTNOTES |
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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.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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