The degree of tissue rescue by PEG-COHb topload transfusion during transient focal cerebral ischemia is substantially greater than that reported with hypervolemic exchange transfusion with αα-crosslinked Hb in rats [1
] or polymeric Hb in mice [6
]. This finding is remarkable because the plasma Hb concentration attained with the 10 ml/kg topload was 0.6 g/dL, which is considerably less than the 2–2.5 g/dL typically attained with 30–40% exchange transfusion of fluids containing 6 g/dL of Hb [19
]. Moreover, increasing the plasma concentration of αα-crosslinked Hb further by increasing the concentration of Hb in the transfusion fluid from 10 g/dL to 20 g/dL produces additional reductions in infarct volume [21
]. These comparisons suggest that PEG-COHb may be superior per mole of heme compared to αα-crosslinked Hb and polymeric Hb in rescuing the brain from stroke, although we cannot exclude differences arising from the exchange transfusion protocol or differences between mouse and rat. Nevertheless, the results indicate that large plasma concentrations of PEGylated COHb are not required to rescue the brain. The theoretical advantage of exchange transfusion is that whole blood viscosity can be reduced by decreasing hematocrit and thereby promote collateral blood flow. The advantage of the topload over the exchange transfusion is that this protocol would be easier to implement in clinical stroke and the 10 ml/kg volume is readily tolerated without producing marked hypervolemic-induced hypertension. Significant hypertension was not observed in the present study.
Rescue by the PEG-COHb did not appear to be related to an increase in blood flow in the ischemic core of cortex as assessed by laser-Doppler flowmetry at a single site. However, it is possible that collateral blood flow was improved in the ischemic border region sufficient to salvage tissue. The viscosity of the PEGylated Hb solution is closer to that of whole blood compared to solutions of crosslinked and polymeric Hb and thus may better maintain endothelial shear stress and associated NO production, which could help maintain dilation of collateral arteries.
The 10-ml/kg topload transfusion of PEG-albumin and PEG-COHb produced approximately 8–10% decreases in hematocrit. This relatively small decrease in hematocrit will have only a small effect on blood viscosity and is unlikely to improve perfusion sufficiently to reduce brain injury by reducing blood viscosity. The lack of a difference in infarct volume between the group transfused with PEG-albumin and the group with no transfusion in this study and in a previous study with greater hemodilution [7
] supports this premise. Moreover, the increase in plasma [Hb] to 0.6 g/dL after PEG-COHb topload was inadequate to offset the decrease in hematocrit. Thus, whole blood [Hb] and arterial O2
content were not increased by PEG-COHb transfusion. However, even in the absence of an increase in arterial O2
content or blood flow, an O2
carrier in the plasma may be capable of enhancing O2
delivery to ischemic tissue for several reasons. First, oxygen solubility in plasma is low and represents a major site of resistance of O2
diffusion between the red cell and mitochondria in the tissue. By carrying a large amount of O2
in the plasma, O2
transport from the red cell membrane to the endothelium is facilitated. In this regard, efficacy of transfusion of Hb polymers during MCAO is lost when the solution primarily contains polymers of molecular weight greater than 14 MDa [7
]. The current bovine PEG-Hb with approximately 8–10 side chains of 5000 molecular weight PEG may represent a good balance of being small enough to have high mobility for facilitating O2
transport in the plasma but large enough to limit extravasation. Second, red cell flux through individual microvessels is heterogeneous and this heterogeneity is amplified under conditions of low perfusion pressure during incomplete ischemia. By delivering O2
through the flow of plasma in capillaries that are red cell deprived, O2
delivery may become more homogenous among capillaries. Third, red cells are particulate and their surface area does not cover the entire surface area of capillary endothelium at any one instant. An O2
carrier in the plasma increases the effective surface area for O2
diffusion. Therefore, a topload infusion of cell-free Hb could invoke several mechanisms of O2
delivery independent of bulk arterial O2
content and macroscopic blood flow.
Another consideration is that the PEG-Hb product was transfused in the carboxy state. Vandergriff et al. [22
] reported that mixing human PEG-COHb with red blood cells resulted in equilibration of the percent of COHb in each compartment at 0.5 h after mixing. In the present study using ~80% COHb in the stock solution of PEG-Hb, the COHb in the plasma Hb was in the 10% range at 0.5 h after transfusion and whole blood COHb was ~2%. Thus, red blood cell Hb and the rest of the body provided a large sink for CO. The residual amount of 2% COHb in whole blood has only a minor effect on oxygen carry capacity. Thus, most of the transfused PEG-COHb can act as an O2
carrier in a relatively short timeframe after transfusion.
In addition, part of the brain protection from ischemia seen with PEG-COHb might be attributed to release of CO. Small molecules that release CO have been shown to exert vasodilatory, anti-inflammatory, and anti-apoptotic properties in a variety of models in the peripheral circulation [23
] and to protect the neonatal brain vasculature from seizure-induced dysfunction [24
]. Moreover, transfusion of red blood cells containing high COHb was shown to be beneficial in limiting microvascular markers of apoptosis after hemorrhagic shock [25
]. Furthermore, combined pretreatment and post-treatment with human PEG-COHb has been shown to protect the heart from coronary artery occlusion in rats [22
]. Thus, the use of PEG-COHb to release a well-defined amount of CO may have potential benefit in ischemic tissue. Future work is required to define the role of CO and its mechanism of action in the protection afforded by PEG-COHb. Interestingly, small amounts of PEG-Hb can protect the brain from ischemia when the PEG-Hb is transfused in the S-nitrosylated form [16
]. Thus, transfusion of PEG-Hb in either the carboxy or S-nitrosylated form may enhance the efficacy in cerebral ischemia.
In summary, the present work provides a proof of principle that PEG-COHb has potential as a therapy in ischemic stroke. Further work is required to determine the therapeutic time window of opportunity for administration, the optimal transfusion protocol and dose, long-term effects on neurobehavior, efficacy in a model of more prolonged ischemia, and a possible role of CO released from the Hb.