Role of 20-HETE in the pial arteriolar constrictor response to decreased hematocrit after exchange transfusion of cell-free polymeric hemoglobin.

The cerebrovascular response to decreases in hematocrit and viscosity depends on accompanying changes in arterial O2 content. This study examines whether 1) the arteriolar dilation seen after exchange transfusion with a 5% albumin solution can be reduced by the K(ATP) channel antagonist glibenclamide (known to inhibit hypoxic dilation), and 2) the arteriolar constriction seen after exchange transfusion with a cell-free hemoglobin polymer to improve O2-carrying capacity can be blocked by inhibitors of the synthesis or vasoconstrictor actions of 20-HETE. In anesthetized rats, decreasing hematocrit by one-third with albumin exchange transfusion dilated pial arterioles (14 +/- 2%; SD), whereas superfusion of the surface of the brain with 10 muM glibenclamide blocked this response (-10 +/- 7%). Exchange transfusion with polymeric hemoglobin decreased the diameter of pial arterioles by 20 +/- 3% without altering arterial pressure. This constrictor response was attenuated by superfusing the surface of the brain with a 20-HETE antagonist, WIT-002 (10 microM; -5 +/- 1%), and was blocked by two chemically dissimilar selective inhibitors of the synthesis of 20-HETE, DDMS (50 microM; 0 +/- 4%) and HET-0016 (1 microM; +6 +/- 4%). The constrictor response to hemoglobin transfusion was not blocked by an inhibitor of nitric oxide (NO) synthase, and the inhibition of the constrictor response by DDMS was not altered by coadministration of the NO synthase inhibitor. We conclude 1) that activation of K(ATP) channels contributes to pial arteriolar dilation during anemia, whereas 2) constriction to polymeric hemoglobin transfusion at reduced hematocrit represents a regulatory response that limits increased O2 transport and that is mediated by increased formation of 20-HETE, rather than by NO scavenging.     

Insensitivity of cerebral oxygen transport to oxygen affinity of hemoglobin-based oxygen carriers

The cerebrovascular effects of exchange transfusion of various cell-free hemoglobins that possess different oxygen affinities are reviewed. Reducing hematocrit by transfusion of a non-oxygen-carrying solution dilates pial arterioles on the brain surface and increases cerebral blood flow to maintain a constant bulk oxygen transport to the brain. In contrast, transfusion of hemoglobins with P50 of 4-34 Torr causes constriction of pial arterioles that offsets the decrease in blood viscosity to maintain cerebral blood flow and oxygen transport. The autoregulatory constriction is dependent on synthesis of 20-HETE from arachidonic acid. This oxygen-dependent reaction is apparently enhanced by facilitated oxygen diffusion from the red cell to the endothelium arising from increased plasma oxygen solubility in the presence of low or high-affinity hemoglobin. Exchange transfusion of recombinant hemoglobin polymers with P50 of 3 and 18 Torr reduces infarct volume from experimental stroke. Cell-free hemoglobins do not require a P50 as high as red blood cell hemoglobin to facilitate oxygen delivery.     

Vascular response to infusions of a nonextravasating hemoglobin polymer.

The clinical utility of cross-linked tetrameric hemoglobin solutions is limited by peripheral vasoconstriction thought to be due to scavenging of nitric oxide. In addition, transfusion of crude preparations of hemoglobin polymers can cause arterial hypertension. We tested the hypothesis that eliminating low-molecular-weight components from the polymer solution would prevent extravasation and its associated pressor response. A zero-link polymer of bovine hemoglobin was developed without chemical linkers left between the tetramers. Transfusion of unprocessed preparations of these polymers in rats resulted in appearance of the polymer in the renal hilar lymph. However, eliminating the low-molecular-weight components with a 300-kDa diafiltration resulted in an average hydrodynamic radius of 250 A and in undetectable levels of polymer in hilar lymph. Exchange transfusion in anesthetized rats and cats and in awake cats produced no increase in arterial pressure. In anesthetized cats, exchange transfusion with an albumin solution reduced hematocrit from 30 to 18%, increased cerebral blood flow, and dilated pial arterioles. In contrast, reducing hematocrit by transfusing the diafiltered polymer did not increase cerebral blood flow as pial arterioles constricted. These results are consistent with the hypothesis that the increase in arterial pressure associated with cell-free hemoglobin transfusion depends on hemoglobin extravasation. Constriction observed in the cerebrovascular bed with a nonextravasating hemoglobin polymer at low hematocrit is presumably a regulatory response to prevent overoxygenation at low blood viscosity.     

Pericardial delivery of omega-3 fatty acid: a novel approach to reducing myocardial infarct sizes and arrhythmias

O2-carrying fluids based on hemoglobin (Hb) are in various stages of clinical trials to determine their suitability as O2-carrying plasma expanders. Polymerized Hb solutions are characterized by their vasoactivity, low O2 affinity, oncotic effect, prolonged shelf life, and stability. Physiological responses to facilitated O2 transport after exchange transfusion with polymerized bovine Hb (PBH) were studied in the hamster window chamber model during acute moderate anemia to determine how PBH affects microvascular perfusion and tissue oxygenation. The anemic state [29% hematocrit (Hct)] was induced by hemodilution with a plasma expander (70 kDa dextran). After hemodilution, animals were randomly assigned to different exchange transfusion groups. Study groups were based on the concentration of PBH used, namely: PBH at 13 g Hb/dl [PBH13], PBH diluted to 8 (PBH8) or 4 (PBH4) g Hb/dl in albumin solution at matching colloidal osmotic pressure (COP), and no PBH (only albumin solution) at matching COP (PBH0). Measurement of systemic parameters, microvascular hemodynamics, capillary perfusion, and intravascular and tissue O2 levels was performed at 18% Hct. Restitution of O2-carrying capacity with PBH13 increased arterial pressure and triggered vasoconstriction, low perfusion, and high peripheral resistance. PBH4 and PBH0 exhibited lower arterial pressures compared with PBH13. Exchange transfused animals with PBH8 and PBH4 better maintained perfusion and functional capillary density than PBH13. Blood gas parameters and acid-base balance were recovered proportional to microvascular perfusion. Arterial O2 tensions were improved with PBH4 and PBH8 by preventing O2 precapillary release and increasing O2 reserve. Further studies to establish PBH optimal dosage, efficacy, safety, and its effect on outcome are indicated before Hb-based O2-carrying blood substitutes are implemented in routine practice.     

Regional cerebral blood flow in cats with cross-linked hemoglobin transfusion during focal cerebral ischemia

The beneficial effect of hemodilution on cerebral blood flow (CBF) during focal cerebral ischemia is mitigated by reduced arterial oxygen content (CaO2). In anesthetized cats subjected to permanent middle cerebral artery occlusion, the time course of regional CBF was evaluated after isovolemic exchange transfusion with either albumin or a tetrameric hemoglobin-based oxygen carrier. The transfusion started 30 min after arterial occlusion. We tested the hypothesis that bulk oxygen transport (CBF x CaO2) to ischemic tissue is increased by hemoglobin transfusion at a hematocrit of 18% compared with albumin-transfused cats at a hematocrit of 18% or control cats at a hematocrit of 30% and equivalent arterial pressure. In the nonischemic hemisphere, CBF increased selectively after albumin transfusion, and oxygen transport was similar among groups. In the ischemic cortex, albumin transfusion increased CBF, but oxygen transport was not increased above that of the control group. Hemoglobin transfusion increased both CBF and oxygen transport in the ischemic cortex above values in the control group, but the increase was delayed until 4 h of ischemia. Consequently, acute injury volume measured at 6 h of ischemia was not significantly attenuated. In contrast to the cortex, CBF in the ischemic caudate nucleus was not substantially increased by either albumin or hemoglobin transfusion. Therefore, in a large animal model of permanent focal ischemia in which transfusion starts 30 min after ischemia, tetrameric cross-linked hemoglobin transfusion can augment oxygen transport to the ischemic cortex, but the increase can be delayed and not necessarily provide protection. Moreover, an end-artery region such as the caudate nucleus is less likely to benefit from hemodilution.     

Role of nitric oxide scavenging in vascular response to cell-free hemoglobin transfusion

Modified Hb solutions have been developed as O(2) carrier transfusion fluids, but of concern is the possibility that increased scavenging of nitric oxide (NO) within the plasma will alter vascular reactivity even if the Hb does not readily extravasate. The effect of decreasing hematocrit from approximately 30% to 18% by an exchange transfusion of a 6% sebacyl cross-linked tetrameric Hb solution on the diameter of pial arterioles possessing tight endothelial junctions was examined through a cranial window in anesthetized cats with and without a NO synthase (NOS) inhibitor. Superfusion of a NOS inhibitor decreased diameter, and subsequent Hb transfusion produced additional constriction that was not different from Hb transfusion alone but was different from the dilation observed by exchange transfusion of an albumin solution after NOS inhibition. In contrast, abluminal application of the cross-linked Hb produced constriction that was attenuated by the NOS inhibitor. Neither abluminal nor intraluminal cross-linked Hb interfered with pial arteriolar dilation to cromakalim, an activator of ATP-sensitive potassium channels. Pial vascular reactivity to hypocapnia and hypercapnia was unaffected by Hb transfusion. Microsphere-determined regional blood flow indicated selective decreases in perfusion after Hb transfusion in the kidney, small intestine, and neurohypophysis, which does not have tight endothelial junctions. Administration of a NOS inhibitor to reduce the basal level of NO available for scavenging before Hb transfusion prevented further decreases in blood flow to these regions compared with NOS inhibition alone. In contrast, blood flow to skeletal and left ventricular muscle increased, and cerebral blood flow was unchanged after Hb transfusion. This cross-linked Hb tetramer is known to appear in renal lymph but not in urine. We conclude that cell-free tetrameric Hb does not scavenge sufficient NO in the plasma space to significantly affect baseline tone in vascular beds with tight endothelial junctions but does produce substantial constriction in beds with porous endothelium. The data support increasing the molecular size of Hb by polymerization or conjugation to limit extravasation in all vascular beds to preserve normal vascular reactivity.     

Role of heme oxygenase-2 in pial arteriolar response to acetylcholine in mice with and without transfusion of cell-free hemoglobin polymers

Carbon monoxide derived from heme oxygenase (HO) may participate in cerebrovascular regulation under specific circumstances. Previous work has shown that HO contributes to feline pial arteriolar dilation to acetylcholine after transfusion of a cell-free polymeric hemoglobin oxygen carrier. The role of constitutive HO2 in the pial arteriolar dilatory response to acetylcholine was determined by using 1) HO2-null mice (HO2-/-), 2) the HO inhibitor tin protoporphyrin IX (SnPPIX), and 3) 4,5,6,7-tetrabromobenzotriazole (TBB), an inhibitor of casein kinase-2 (CK2)-dependent phosphorylation of HO2. In anesthetized mice, superfusion of a cranial window with SnPPIX decreased arteriolar dilation produced by 10 microM acetylcholine by 51%. After partial polymeric hemoglobin exchange transfusion, the acetylcholine response was normal but was reduced 72% by SnPPIX and 95% by TBB. In HO2-/- mice, the acetylcholine response was modestly reduced by 14% compared with control mice and was unaffected by SnPPIX. After hemoglobin transfusion in HO2-/- mice, acetylcholine responses were also unaffected by SnPPIX and TBB. In contrast, nitric oxide synthase inhibition completely blocked the acetylcholine responses in hemoglobin-transfused HO2-/- mice. We conclude 1) that HO2 activity partially contributes to acetylcholine-induced pial arteriolar dilation in mice, 2) that this contribution is augmented in the presence of a plasma-based hemoglobin polymer and appears to depend on a CK2 kinase mechanism, 3) that nitric oxide synthase activity rather than HO1 activity contributes to the acetylcholine reactivity in HO2-/- mice, and 4) that plasma-based polymeric hemoglobin does not scavenge all of the nitric oxide generated by cerebrovascular acetylcholine stimulation.     

Dependence of acetylcholine and ADP dilation of pial arterioles on heme oxygenase after transfusion of cell-free polymeric hemoglobin

Polymers of cell-free hemoglobin have been designed for clinical use as oxygen carriers, but limited information is available regarding their effects on vascular regulation. We tested the hypothesis that the contribution of heme oxygenase (HO) to acetylcholine-evoked dilation of pial arterioles is upregulated 2 days after polymeric hemoglobin transfusion. Dilator responses to acetylcholine measured by intravital microscopy in anesthetized cats were blocked by superfusion of the HO inhibitor tin protoporphyrin-IX (SnPPIX) in a group that had undergone exchange transfusion with hemoglobin 2 days earlier but not in surgical sham and albumin-transfused groups. However, immunoblots from cortical brain homogenates did not reveal changes in expression of the inducible isoform HO1 or the constitutive isoform HO2 in the hemoglobin-transfused group. To test whether the inhibitory effect of SnPPIX was present acutely after hemoglobin transfusion, responses were measured within an hour of completion of the exchange transfusion. In control and albumin-transfused groups, acetylcholine responses were unaffected by SnPPIX but were blocked by addition of the nitric oxide synthase inhibitor N(omega)-nitro-l-arginine (l-NNA) to the superfusate. In hemoglobin-transfused groups, the acetylcholine response was blocked by either SnPPIX or l-NNA alone. The effect of another HO inhibitor, chromium mesoporphyrin (CrMP), was tested on ADP, another endothelial-dependent dilator, in anesthetized rats. Pial arteriolar dilation to ADP was unaffected by CrMP in controls but was attenuated 62% by CrMP in rats transfused with hemoglobin. It is concluded that 1) polymeric hemoglobin transfusion acutely upregulates the contribution of HO to acetylcholine-induced dilation of pial arterioles in cats, 2) this upregulation persists 2 days after transfusion when 95% of the hemoglobin is cleared from the circulation, and 3) this acute upregulation of HO signaling is ubiquitous in that similar effects were observed with a different endothelial-dependent agonist (i.e., ADP) in a another species (rat).     

Comparison of PEG-modified albumin and hemoglobin in extreme hemodilution in the rat.

We have reported a new polyethylene glycol (PEG)-modified, hemoglobin-based O2 carrier (MP4) with novel properties, including a large molecular excluded volume and low PO2 necessary to obtain 50% O2 (approximately 6 Torr). To evaluate the ability of MP4 to transport O2, we compared it with PEG-modified albumin (MPA) using the identical chemistry of attachment of PEG chains. The resulting solutions were well matched with respect to all physical properties except that MP4 is an O2 carrier, whereas MPA is not. An additional solution, 10% pentastarch, was matched with the PEG-modified proteins with regard to oncotic activity and viscosity but does not contain PEG. The model used to evaluate O2 transport was continuous exchange transfusion in the rat until the hematocrit was virtually unmeasurable. Objective end points included survival and the onset of anaerobic metabolism, signaled by acid-base derangement and accumulation of lactic acid. Continuous exchange transfusion of 2.5 blood volumes in rats (n=5 in each treatment group) was carried out over 60 min, such that the final hematocrit was between 0 and 5% in all animals. Animals were observed for an additional 70 min, when survivors were killed. Overall survival for the MP4 animals was 100%; no animal that received either pentastarch or MPA survived. The hematocrit at which lactic acid began to rise was approximately 14.8% in both pentastarch and MPA animals and 7.4% in the animals that received MP4. In all groups, the total hemoglobin was approximately 5 g/dl at this point. We conclude that, despite its low PO2 necessary to obtain 50% O2, MP4 effectively substitutes for red blood cell hemoglobin in its ability to oxygenate tissues in extreme hemodilution.     

Increased tissue PO2 and decreased O2 delivery and consumption after 80% exchange transfusion with polymerized hemoglobin

The O2-carrying blood substitute based on polymerized bovine hemoglobin (PBH) was used to determine efficacy in maintaining tissue Po2 after an 80% isovolemic blood exchange leading to a hematocrit of 19% [5.4 g Hb/dl from red blood cells (RBCs) and 6.3 g Hb/dl from PBH]. Effects were studied in terms of O2 delivery, O2 extraction, and tissue Po2 at the microcirculatory level at 1, 12, and 24 h after exchange transfusion in awake hamsters prepared with a window chamber model. At 1 h after exchange, arteriolar and venular diameters were decreased compared with baseline. Arteriolar diameter did not fully recover at 12 h after exchange, but venular diameter returned to normal. At 24 h after exchange, arteriolar and venular diameters were not different from baseline. Combining diameter and flow velocity data allowed us to calculate arteriolar and venular flows. At 1 h after exchange, arteriolar and venular flow was reduced compared with baseline. Arteriolar flow was lower at 12 h after exchange and recovered after 24 h. The number of capillaries with RBC passage [functional capillary density (FCD)] at 1 h after exchange with PBH was significantly lower than baseline. FCD remained decreased at 12 h; at 24 h after exchange transfusion, FCD was fully recovered. Tissue Po2 was maximal at 1 h after exchange and decreased progressively at 12 and 24 h after exchange. O2 release to the tissue was minimal at 1 h and increased at 12 and 24 h after exchange. These results suggest the impairment of tissue O2 metabolism after introduction of PBH into the circulation, which is mitigated as PBH concentration declines.     

A compartmental model for oxygen transport in brain microcirculation in the presence of blood substitutes

A compartmental model is developed for oxygen (O(2)) transport in brain microcirculation in the presence of blood substitutes (hemoglobin-based oxygen carriers). The cerebrovascular bed is represented as a series of vascular compartments, on the basis of diameters, surrounded by a tissue compartment. A mixture of red blood cells (RBC) and plasma/extracellular hemoglobin solution flows through the vascular bed from the arterioles through the capillaries to the venules. Oxygen is transported by convection in the vascular compartments and by diffusion in the surrounding tissue where it is utilized. Intravascular resistance and the diffusive loss of oxygen from the arterioles to the tissue are incorporated in the model. The model predicts that most of the O(2) transport occurs at the level of capillaries. Results computed from the present model in the presence of hemoglobin-based oxygen carriers are consistent with those obtained from the earlier validated model (Sharan et al., 1989, 1998a) on oxygen transport in brain circulation in the absence of extracellular hemoglobin. We have found that: (a) precapillary PO(2) gradients increase as PO(2) in the arterial blood increases, P(50 p) (oxygen tension at 50% saturation of hemoglobin with O(2) in plasma) decreases, i.e. O(2) affinity of the extracellular hemoglobin is increased, the flow rate of the mixture decreases, hematocrit decreases at constant flow, metabolic rate increases, and intravascular transport resistance in the arterioles is neglected; (b) precapillary PO(2) gradients are not sensitive to (i) intracapillary transport resistance, (ii) cooperativity (n(p)) of hemoglobin with oxygen in plasma, (iii) hemoglobin concentration in the plasma and (iv) hematocrit when accounting for viscosity variation in the flow; (c) tissue PO(2) is not sensitive to the variation of intravascular transport resistance in the arterioles. We also found that tissue PO(2) is a non-monotonic function of the Hill coefficient n(p) for the extracellular hemoglobin with a maximum occurring when n(p) equals the blood Hill coefficient. The results of the computations give estimates of the magnitudes of the increases in tissue PO(2) as arterial PO(2) increases,P(50 p) increases, flow rate increases, hematocrit increases, hemoglobin concentration in the plasma increases, metabolic rate decreases, the capillary mass transfer coefficient increases or the intracapillary transport resistance decreases.     

The effects of erythrocythemia on blood viscosity,maximal systemic oxygen transport capacity and maximal rates of oxygen consumption in an amphibian

Graded erythrocythemia was induced by isovolemic loading of packed red blood cells in the toad, Bufo marinus. Blood viscosity, hematocrit, hemoglobin concentration, maximal aortic blood flow rate and maximal rates of oxygen consumption were determined after each load. Blood viscosity was related to hematocrit in the expected exponential manner; ln eta = 0.43 + 0.035 Hct. Maximal blood flow rates in the dorsal aorta were inversely proportional to blood viscosity and fit predictions of the Poiseuille-Hagen flow formula. The effect of increased blood viscosity was to reduce aortic pulse volume, but not maximal heart rate. Maximal systemic oxygen transport capacity (aortic blood flow rate X hemoglobin concentration X O2 binding capacity of hemoglobin) was linearly correlated with the maximal rate of oxygen consumption. These date indicate that optimal hematocrit theory is applicable for maximal blood flow rates in vivo, and that systemic oxygen transport is the primary limitation to aerial VO2 max in amphibians.     

beta-Adrenoceptor and nNOS-derived NO interactions modulate hypoglycemic pial arteriolar dilation in rats

We examined the relative contributions from nitric oxide (NO) and catecholaminergic pathways in promoting cerebral arteriolar dilation during hypoglycemia (plasma glucose congruent with 1.4 mM). To that end, we monitored the effects of beta-adrenoceptor (beta-AR) blockade with propranolol (Pro, 1.5 mg/kg iv), neuronal nitric oxide synthase (nNOS) inhibition with 7-nitroindazole (7-NI, 40 mg/kg ip) or ARR-17477 (300 microM, via topical application), or combined intravenous Pro + 7-NI or ARR-17477 on pial arteriolar diameter changes in anesthetized rats subjected to insulin-induced hypoglycemia. Additional experiments, employing topically applied TTX (1 microM), addressed the possibility that the pial arteriolar response to hypoglycemia required neuronal transmission. Separately, Pro and 7-NI elicited modest but statistically insignificant 10-20% reductions in the normal ~40% increase in arteriolar diameter accompanying hypoglycemia. However, combined Pro-7-NI was accompanied by a >80% reduction in the hypoglycemia-induced dilation. On the other hand, the combination of intravenous Pro and topical ARR-17477 did not affect the hypoglycemia response. In the presence of TTX, the pial arteriolar response to hypoglycemia was lost completely. These results suggest that 1) beta-ARs and nNOS-derived NO interact in contributing to hypoglycemia-induced pial arteriolar dilation; 2) the interaction does not occur in the vicinity of the arteriole; and 3) the vasodilating signal is transmitted via a neuronal pathway.     

Effects of estrogen on cerebral blood flow and pial microvasculature in rabbits

We tested the hypothesis that intracarotid estrogen infusion increases cerebral blood flow (CBF) in a concentration-dependent manner and direct application of estrogen on pial arterioles yields estrogen receptor-mediated vasodilation. Rabbits of both genders were infused with estrogen via a branch of the carotid artery. Estrogen doses of 20 or 0.05 microg. ml(-1). min(-1) were used to achieve supraphysiological or physiological plasma estrogen levels, respectively. CBF and cerebral vascular resistance were determined at baseline, during the infusion, and 60-min postinfusion, and effects on pial diameter were assessed via a cranial window. Pial arteriolar response to estrogen alone and to estrogen after administration of tamoxifen (10(-7)), an antiestrogen drug that binds to both known estrogen receptor subtypes, was tested. No gender differences were observed; therefore, data were combined for both males and females. Systemic estrogen infusion did not increase regional CBF. Estradiol dilated pial arteries only at concentrations ranging from 10(-4)-10(-7) M (P < or = 0.05). Pretreatment with tamoxifen alone had no effect on arteriolar diameter but inhibited estrogen-induced vasodilation (P < 0.001). Our data suggest that estrogen does not increase CBF under steady-state conditions in rabbits. In the pial circulation, topically applied estradiol at micromolar concentrations dilates vessels. The onset is rapid and dependent on estrogen receptor activation.     

Effect of hematocrit on cerebral blood flow with induced polycythemia

Cerebral blood flow (CBF) is lowered during polycythemia. Whether this fall is due to an increase in red blood cell concentration (Hct) or to an increase in arterial O2 content (Cao2) is controversial. We examined the independent effects of Hct and Cao2 on CBF as Hct was raised from 30 to 55% in anesthetized 1- to 7-day-old sheep. CBF was measured by the radiolabeled microsphere technique before and after isovolemic exchange transfusion with either oxyhemoglobin-containing erythrocytes (in 5 control animals) or with methemoglobin-containing erythrocytes (in 9 experimental animals). Following exchange transfusion in the control animals, Hct rose (30 +/- 1 vs. 55 +/- 1%, mean +/- SE), Cao2 increased (15.1 +/- 0.8 vs. 26.7 +/- 0.9 vol%), and CBF fell (66 +/- 9 vs. 35 +/- 5 ml X min-1 X 100 g-1). Because the fall in CBF was proportionate to the rise in Cao2, cerebral O2 transport (CBF X Cao2) was unchanged. Following exchange transfusion in the experimental animals, Hct rose (32 +/- 1 vs. 55 +/- 1%) but Cao2 did not change. Nevertheless, CBF still fell (73 +/- 4 vs. 48 +/- 2 ml X min-1 X 100 g-1) and, as a result, cerebral O2 transport also fell. The latter cannot be attributed to a fall in cerebral O2 uptake, as cerebral O2 uptake was unaffected during each of these conditions. Comparison of the two groups of animals showed that approximately 60% of the fall in CBF may be attributed to the increase in red cell concentration alone. It is probable that this effect is due largely to changes in blood viscosity.     

A quantitative framework for the design of acellular hemoglobins as blood substitutes: implications of dynamic flow conditions

The delivery of oxygen to tissue by cell-free carriers eliminates intraluminal barriers associated with red blood cells. This is important in arterioles, since arteriolar tone controls capillary perfusion. We describe a mathematical model for O(2) transport by hemoglobin solutions and red blood cells flowing through arteriolar-sized tubes to optimize values of p50, Hill number, hemoglobin molecular diffusivity and concentration. Oxygen release is evaluated by including an extra-luminal resistance term to reflect tissue oxygen consumption. For low consumption (i.e., high resistance to O(2) release) a hemoglobin solution with p50=15 mmHg, n=1, D(HBO2)=3 x 10(-7) cm(2)/s delivers O(2) at a rate similar to that of red blood cells. For high consumption, the p50 must be decreased to 5 mmHg. The model predicts that regardless of size, hemoglobin solutions with higher p50 will present excess O(2) to arteriolar walls. Oversupply of O(2) to arteriolar walls may cause constriction and paradoxically reduced capillary perfusion.     

Plasma viscosity and cerebral blood flow

We hypothesized that the response of cerebral blood flow (CBF) to changing viscosity would be dependent on "baseline" CBF, with a greater influence of viscosity during high-flow conditions. Plasma viscosity was adjusted to 1.0 or 3.0 cP in rats by exchange transfusion with red blood cells diluted in lactated Ringer solution or with dextran. Cortical CBF was measured by H(2) clearance. Two groups of animals remained normoxic and normocarbic and served as controls. Other groups were made anemic, hypercapnic, or hypoxic to increase CBF. Under baseline conditions before intervention, CBF did not differ between groups and averaged 49.4 +/- 10.2 ml. 100 g(-1). min(-1) (+/-SD). In control animals, changing plasma viscosity to 1. 0 or 3.0 cP resulted in CBF of 55.9 +/- 8.6 and 42.5 +/- 12.7 ml. 100 g(-1). min(-1), respectively (not significant). During hemodilution, hypercapnia, and hypoxia with a plasma viscosity of 1. 0 cP, CBF varied from 98 to 115 ml. 100 g(-1). min(-1). When plasma viscosity was 3.0 cP during hemodilution, hypercapnia, and hypoxia, CBF ranged from 56 to 58 ml. 100 g(-1). min(-1) and was significantly reduced in each case (P < 0.05). These results support the hypothesis that viscosity has a greater role in regulation of CBF when CBF is increased. In addition, because CBF more closely followed changes in plasma viscosity (rather than whole blood viscosity), we believe that plasma viscosity may be the more important factor in controlling CBF.     

Arachidonic acid- and prostaglandin E2-induced cerebral vasodilation is mediated by carbon monoxide, independent of reactive oxygen species in piglets

Arachidonic acid (AA) and prostaglandin (PG) E(2) stimulate carbon monoxide (CO) production, and AA metabolism is known to be associated with the generation of reactive oxygen species (ROS). This study was conducted to address the hypothesis that CO and/or ROS mediate cerebrovascular dilation in newborn pigs. Experiments were performed on anesthetized newborn pigs with closed cranial windows. Different concentrations of AA (10(-8)-10(-6) M), PGE(2) (10(-8)-10(-6) M), iloprost (10(-8)-10(-6) M), and their vehicle (artificial cerebrospinal fluid) were given. Piglets with PGE(2) and iloprost received indomethacin (5 mg/kg iv) to inhibit cyclooxygenase. AA, PGE(2), and iloprost caused concentration-dependent increases in pial arteriolar diameter. The effects of both AA and PGE(2) in producing cerebral vascular dilation and associated CO production were blocked by the heme oxygenase inhibitor chromium mesoporphyrin (2 × 10(-5) M), but not by the prostacyclin analog, iloprost. ROS inhibitor tempol (SOD mimetic) (1 × 10(-5) M) and the H(2)O(2) scavenger catalase (1,000 U/ml) also do not block these vasodilator effects of AA and PGE(2). Heme-L-lysinate-induced cerebrovascular dilation and CO production was blocked by chromium mesoporphyrin. Hypoxanthine plus xanthine oxidase, a combination that is known to generate ROS, caused pial arteriolar dilation and CO production that was inhibited by tempol and catalase. These data suggest that AA- and PGE(2)-induced cerebral vascular dilation is mediated by CO, independent of ROS.     

Targeted O2 delivery by low-P50 hemoglobin: a new basis for O2 therapeutics

To assess O2 delivery to tissue by a new surface-modified, polyethylene glycol-conjugated human hemoglobin [MP4; Po2 at 50% saturation of hemoglobin (P50); 5.4 mmHg], we studied microcirculatory hemodynamics and O2 release in golden Syrian hamsters hemodiluted with MP4 or polymerized bovine hemoglobin (PolyBvHb; P50 54.2 mmHg). Comparisons were made with the animals' hemodiluted blood with a non-O2 carrying plasma expander with similar solution properties (Dextran-70). Systemic hemodynamics (arterial blood pressure and heart rate) and acid-base parameters were not correlated with microhemodynamics (arteriolar and venular diameter, red blood cell velocity, and flow). Microscopic measurements of Po2 and the O2 equilibrium curves permitted analysis of O2 release in precapillary and capillary vessels by red blood cells and plasma hemoglobin separately. No significant differences between the groups of animals with respect to arteriolar diameter, flow, or flow velocity were observed, but the functional capillary density was significantly higher in the MP4-treated animals (67%) compared with PolyBvHb-treated animals (37%; P < 0.05) or dextran-treated animals (53%). In the PolyBvHb-treated animals, predominant O2 release (both red blood cells and plasma hemoglobin) occurred in precapillary vessels, whereas in MP4 animals most of the O2 was released from both red blood cells and plasma hemoglobin in capillaries. Base excess correlated directly with capillary O2 release but not systemic O2 content or total O2 release. Higher O2 extraction of both red blood cell and plasma hemoglobin in capillaries represents a new mechanism of action of cell-free hemoglobin. High O2 affinity appears to be an important property for cell-free hemoglobin solutions.     

Pial arteriole dilation during somatosensory stimulation is not mediated by an increase in CSF metabolites

Pial arterioles supplying the hindlimb somatosensory cortex dilate in response to contralateral sciatic nerve stimulation. The mechanism of this pial vasodilation is not well understood. One possibility is that vasoactive metabolites released during brain activation may diffuse to subarachnoid cerebrospinal fluid (CSF) to dilate pial vessels. To test this hypothesis, we implanted closed cranial windows in rats and measured pial arteriolar dilation to sciatic nerve stimulation during constant rate superfusion of the pial surface with artificial CSF. We reason that flushing the pial surface with CSF should quickly dissipate vasoactive substances and prevent these substances from dilating pial arterioles. CSF flow (1 and 1.5 ml/min) significantly reduced pial arteriole dilation induced by 5% CO2 inhalation, but the same flow rates did not affect dilator responses to sciatic nerve stimulation. We conclude that brain-to-CSF diffusion of vasoactive metabolites does not play a significant role in the dilation of pial arterioles during somatosensory activity.