Cerebrovascular response to decreased hematocrit: effect of cell-free hemoglobin,plasma viscosity,and CO2

The effect of transfusing a nonextravasating, zero-link polymer of cell-free hemoglobin on pial arteriolar diameter, cerebral blood flow (CBF), and O2 transport (CBF x arterial O2 content) was compared with that of transfusing an albumin solution at equivalent reductions in hematocrit (approximately 19%) in anesthetized cats. The influence of viscosity was assessed by coinfusion of a high-viscosity solution of polyvinylpyrrolidone (PVP), which increased plasma viscosity two- to threefold. Exchange transfusion of a 5% albumin solution resulted in pial arteriolar dilation, increased CBF, and unchanged O2 transport, whereas there were no significant changes over time in a control group. Exchange transfusion of a 12% polymeric hemoglobin solution resulted in pial arteriolar constriction and unchanged CBF and O2 transport. Coinfusion of PVP with albumin produced pial arteriolar dilation that was similar to that obtained with transfusion of albumin alone. In contrast, coinfusion of PVP with hemoglobin converted the constrictor response to a dilator response that prevented a decrease in CBF. Pial arteriolar dilation to hypercapnia was unimpaired in groups transfused with albumin or hemoglobin alone but was attenuated in the largest vessels in albumin and hemoglobin groups coinfused with PVP. Unexpectedly, hypocapnic vasoconstriction was blunted in all groups after transfusion of albumin or hemoglobin alone or with PVP. We conclude that 1) the increase in arteriolar diameter after albumin transfusion represents a compensatory response that prevents decreased O2 transport at reduced O2-carrying capacity, 2) the decrease in diameter associated with near-normal O2-carrying capacity after cell-free polymeric hemoglobin transfusion represents a compensatory mechanism that prevents increased O2 transport at reduced blood viscosity, 3) pial arterioles are capable of dilating to an increase in plasma viscosity when hemoglobin is present in the plasma, 4) decreasing hematocrit does not impair pial arteriolar dilation to hypercapnia unless plasma viscosity is increased, and 5) pial arteriolar constriction to hypocapnia is impaired at reduced hematocrit independently of O2-carrying capacity.     

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.     

Changes in cerebral free fatty acids and triacylglycerols in focal cerebral ischemia

1. Focal cerebral ischemia was induced in anesthetized rats by occluding the stem of the proximal middle cerebral artery. 2. The levels of free fatty acids, such as stearic and arachidonic acids, in the ischemic cerebral cortex increased progressively until 60 min after occlusion, but thereafter they decreased rapidly. 3. In contrast to the time-dependent changes in free fatty acids, the levels of triacylglycerol (TAG) in the ischemic cerebral cortex continued to increase for 120 min after occlusion. Increases in TAG-palmitate, -stearate and -arachidonate accounted for the increase in the triacylglycerol level. 4. The pattern of the lipid changes in focal cerebral ischemia differs from those reported in bilateral diffuse cerebral ischemia induced by arterial occlusion or in decapitation ischemia.     

A recombinant polymeric hemoglobin with conformational,functional, and physiological characteristics of an in vivo O2 transporter

With the objective of developing a recombinant oxygen carrier suitable for therapeutic applications, we have employed an Escherichia coli expression system to synthesize in high-yield hemoglobin (Hb) Minotaur, containing alpha-human and beta-bovine chains. Polymerization of Hb Minotaur through S-S intermolecular cross-linking was obtained by introducing a Cys at position beta9 and substituting the naturally occurring Cys. This homogeneous polymer, Hb Polytaur, has a molecular mass of approximately 500 kDa and was resistant toward reducing agents present in blood. In mice, the circulating half-time (3 h) was fivefold greater than adult human Hb (HbA). The half-time of autooxidation measured in blood (46 h) exceeded the circulating retention time. Hypervolemic exchange transfusion resulted in increased arterial blood pressure similar to that with albumin. The increase in pressure was less than that obtained by transfusion of cross-linked tetrameric Hb known to undergo renovascular extravasation. The nitric oxide reactivity of Hb Polytaur was similar to HbA, suggesting that the diminished pressor response to Hb Polytaur was probably related to diminished extravasation. Transfusion of 3% Hb Polytaur during focal cerebral ischemia reduced infarct volume by 22%. Therefore, site-specific Cys insertion on the Hb surface results in uniform size polymers that do not produce the large pressor response seen with tetrameric Hb. Polymerization maintains physiologically relevant oxygen and heme affinity, stability toward denaturation and oxidation, and effective oxygen delivery as indicated by reduced cerebral ischemic damage.     

Salvage of focal cerebral ischemic damage by transfusion of high O2-affinity recombinant hemoglobin polymers in mouse.

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 alpha-subunit and the bovine beta-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 approximately 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.     

Effect of hemodilution on RBC velocity, supply rate, and hematocrit in the cerebral capillary network.

The effect of isovolemic hemodilution on the circulation of red blood cells (RBCs) in the cerebrocortical capillary network was studied by intravital videomicroscopy with use of a closed-cranial-window technique in the rat. Velocity and supply rate of RBCs were measured by tracking the movement and counting the number of fluorescently labeled cells. Arterial blood was withdrawn in increments of 2 ml and replaced by serum albumin. Arterial blood pressure was maintained constant with an infusion of methoxamine. Both velocity and supply rate of RBCs increased, by approximately equal amounts, as arterial hematocrit was reduced from 44 to 15%. The maximum increase in RBC velocity was 4.6 and in RBC supply rate was 5.2 times the baseline value. Calculated lineal density of RBC, an index of capillary hematocrit, did not change with hemodilution. The results suggest that RBC flow and oxygen supply in the cerebral capillary network are maintained during isovolemic hemodilution. The "optimal hematocrit" is as low as 15%.     

Changes in oxygen tension in the cerebral cortex during hemodilution

Wistar rats were subjected to gradual blood replacement with 7% albumin (hemodilution). Hematocrit and mean arterial pressure were measured periodically. Polarographic platinum microelectrodes with a tip 3-8 microns in diameter were used to study variation of oxygen tension (pO2) in the brain cortex during hemodilution. Some areas showed a significant decrease in the brain pO2 after hematocrit dropped to 30%. In animals with an initially low pO2 (13.1 +/- 1.7 mm Hg), this parameter decreased more slowly than in rats with a higher basic pO2 (24.5 +/- 1.7 mm Hg).     

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.     

Oxygen delivery at high blood viscosity and decreased arterial oxygen content to brains of conscious rats

We addressed the question to which extent cerebral blood flow (CBF) is maintained when, in addition to a high blood viscosity (Bvis) arterial oxygen content (CaO2) is gradually decreased. CaO2) was decreased by hemodilution to hematocrits (Hct) of 30, 22, 19, and 15% in two groups. One group received blood replacement (BR) only and served as the control. The second group received an additional high viscosity solution of polyvinylpyrrolidone (BR/PVP). Bvis was reduced in the BR group and was doubled in the BR/PVP. Despite different Bvis, CBF did not differ between BR and BR/PVP rats at Hct values of 30 and 22%, indicating a complete vascular compensation of the increased Bvis at decreased CaO2. At an Hct of 19%, local cerebral blood flow (LCBF) in some brain structures was lower in BR/PVP rats than in BR rats. At the lowest Hct of 15%, LCBF of 15 brain structures and mean CBF were reduced in BR/PVP. The resulting decrease in cerebral oxygen delivery in the BR/PVP group indicates a global loss of vascular compensation. We concluded that vasodilating mechanisms compensated for Bvis increases thereby maintaining constant cerebral oxygen delivery. Compensatory mechanisms were exhausted at a Hct of 19% and lower as indicated by the reduction of CBF and cerebral oxygen delivery.     

Perivascular nitric oxide and superoxide in neonatal cerebral hypoxia-ischemia

Decreased cerebral blood flow (CBF) has been observed following the resuscitation from neonatal hypoxic-ischemic injury, but its mechanism is not known. We address the hypothesis that reduced CBF is due to a change in nitric oxide (NO) and superoxide anion O(2)(-) balance secondary to endothelial NO synthase (eNOS) uncoupling with vascular injury. Wistar rats (7 day old) were subjected to cerebral hypoxia-ischemia by unilateral carotid occlusion under isoflurane anesthesia followed by hypoxia with hyperoxic or normoxic resuscitation. Expired CO(2) was determined during the period of hyperoxic or normoxic resuscitation. Laser-Doppler flowmetry was used with isoflurane anesthesia to monitor CBF, and cerebral perivascular NO and O(2)(-) were determined using fluorescent dyes with fluorescence microscopy. The effect of tetrahydrobiopterin supplementation on each of these measurements and the effect of apocynin and N(omega)-nitro-L-arginine methyl ester (L-NAME) administration on NO and O(2)(-) were determined. As a result, CBF in the ischemic cortex declined following the onset of resuscitation with 100% O(2) (hyperoxic resuscitation) but not room air (normoxic resuscitation). Expired CO(2) was decreased at the onset of resuscitation, but recovery was the same in normoxic and hyperoxic resuscitated groups. Perivascular NO-induced fluorescence intensity declined, and O(2)(-)-induced fluorescence increased in the ischemic cortex after hyperoxic resuscitation up to 24 h postischemia. L-NAME treatment reduced O(2)(-) relative to the nonischemic cortex. Apocynin treatment increased NO and reduced O(2)(-) relative to the nonischemic cortex. The administration of tetrahydrobiopterin following the injury increased perivascular NO, reduced perivascular O(2)(-), and increased CBF during hyperoxic resuscitation. These results demonstrate that reduced CBF follows hyperoxic resuscitation but not normoxic resuscitation after neonatal hypoxic-ischemic injury, accompanied by a reduction in perivascular production of NO and an increase in O(2)(-). The finding that tetrahydrobiopterin, apocynin, and L-NAME normalized radical production suggests that the uncoupling of perivascular NOS, probably eNOS, due to acquired relative tetrahydrobiopterin deficiency occurs after neonatal hypoxic-ischemic brain injury. It appears that both NOS uncoupling and the activation of NADPH oxidase participate in the changes of reactive oxygen concentrations seen in cerebral hypoxic-ischemic injury.     

Improvement in Regional CBF by L-Serine Contributes to Its Neuroprotective Effect in Rats after Focal Cerebral Ischemia

To investigate the mechanisms underlying the neuroprotective effect of L-serine, permanent focal cerebral ischemia was induced by occlusion of the middle cerebral artery while monitoring cerebral blood flow (CBF). Rats were divided into control and L-serine-treated groups after middle cerebral artery occlusion. The neurological deficit score and brain infarct volume were assessed. Nissl staining was used to quantify the cortical injury. L-serine and D-serine levels in the ischemic cortex were analyzed with high performance liquid chromatography. We found that L-serine treatment: 1) reduced the neurological deficit score, infarct volume and cortical neuron loss in a dose-dependent manner; 2) improved CBF in the cortex, and this effect was inhibited in the presence of apamin plus charybdotoxin while the alleviation of both neurological deficit score and infarct volume was blocked; and 3) increased the amount of L-serine and D-serine in the cortex, and inhibition of the conversion of L-serine into D-serine by aminooxyacetic acid did not affect the reduction of neurological deficit score and infarct volume by L-serine. In conclusion, improvement in regional CBF by L-serine may contribute to its neuroprotective effect on the ischemic brain, potentially through vasodilation which is mediated by the small- and intermediate-conductance Ca²⁺-activated K⁺ channels on the cerebral blood vessel endothelium.     

EFFECT OF OLIGEMIA ON REGIONAL METABOLITE LEVELS IN CAT BRAIN

Abstract— Incomplete cerebral ischemia (oligemia) was produced in cat by carotid occlusion combined with arterial hypotension. Lowering arterial pressure to 50–60 Torr for 20 min caused marked alterations of the ATP, phosphocreatine, and lactate content of subcortical white matter. In contrast, metabolite levels in cerebral cortex and caudate nucleus were only moderately perturbed from control values. More severe oligemia resulted when arterial pressure was lowered to 30 Torr for 20 min following carotid occlusion. Metabolite levels in cortex, caudate nucleus, and white matter were greatly altered from control. In the gray matter there was regional heterogeneity of metabolic alteration, as evidenced from the pattern of NADH tissue fluorescence. The cortex contained micro-patches (0.1mm) of increased NADH, which frequently exhibited a columnar orientation.
These findings demonstrate two distinct types of cerebral inhomogeneity of metabolic failure with reduced blood flow; white matter fails before gray matter, and there is micro-heterogeneity of metabolic failure in the gray matter.     

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.     

Hemodilutional anemia is associated with increased cerebral neuronal nitric oxide synthase gene expression.

Severe hemodilutional anemia may reduce cerebral oxygen delivery, resulting in cerebral tissue hypoxia. Increased nitric oxide synthase (NOS) expression has been identified following cerebral hypoxia and may contribute to the compensatory increase in cerebral blood flow (CBF) observed after hypoxia and anemia. However, changes in cerebral NOS gene expression have not been reported after acute anemia. This study tests the hypothesis that acute hemodilutional anemia causes cerebral tissue hypoxia, triggering changes in cerebral NOS gene expression. Anesthetized rats underwent hemodilution when 30 ml/kg of blood were exchanged with pentastarch, resulting in a final hemoglobin concentration of 51.0 +/- 1.2 g/l (n = 7 rats). Caudate tissue oxygen tension (Pbr(O(2))) decreased transiently from 17.3 +/- 4.1 to 14.4 +/- 4.1 Torr (P < 0.05), before returning to baseline after approximately 20 min. An increase in CBF may have contributed to restoring Pbr(O(2)) by improving cerebral tissue oxygen delivery. An increase in neuronal NOS (nNOS) mRNA was detected by RT-PCR in the cerebral cortex of anemic rats after 3 h (P < 0.05, n = 5). A similar response was observed after exposure to hypoxia. By contrast, no increases in mRNA for endothelial NOS or interleukin-1beta were observed after anemia or hypoxia. Hemodilutional anemia caused an acute reduction in Pbr(O(2)) and an increase in cerebral cortical nNOS mRNA, supporting a role for nNOS in the physiological response to acute anemia.     

Reversibility of Nimodipine Binding to Brain in Transient Cerebral Ischemia

Using autoradiography, we have measured the in vivo binding of [3H]nimodipine to brain in a rat model of reversible cerebral ischemia. Ischemia was induced by simultaneous occlusion of the middle cerebral artery (MCA) and ipsilateral common carotid artery by microaneurysm clips. Rats were studied after 15 min of ischemia (ischemic group) or after 45 min of reperfusion following 15 min of ischemia (reperfused group). Regional cerebral blood flow (CBF) was determined autoradiographically using [14C]iodoantipyrine in both ischemic (n = 6) and reperfused (n = 6) groups. During ischemia blood flow in the territory of the MCA was depressed and recovered to normal only in the distal territory of the MCA following reperfusion. [3H]Nimodipine binding in the ischemic group (n = 12) was elevated in ischemic brain regions and declined significantly (p < 0.01) in these regions in the reperfused group (n = 11). The ratio of the volume of cortex showing increased binding to the total volume of the forebrain was 0.113 +/- 0.025 (mean +/- SD) in the ischemic group and declined to 0.080 +/- 0.027 following reperfusion (p < 0.005). In general, infarct was only observed in regions showing persistent elevation of nimodipine binding following reperfusion as determined by histology performed in a separate group of rats (n = 8) after 24 h of reperfusion. We conclude that increased nimodipine binding to ischemic tissue is initially reversible with prompt reestablishment of CBF and is a sensitive indicator of early and reversible ischemia-induced cerebral dysfunction.     

Alteration in brain proteins following occlusion of the middle cerebral artery in rat.

The effects of permanent focal ischemia on specific proteins of the cerebral hemisphere were studied by unilateral occlusion of the middle cerebral artery (MCAO) in rat. Brain proteins were prepared 72 h after the occlusion and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were identified by their interaction with rabbit antibodies against rat serum proteins and anti-transferrin antibodies. SDS-PAGE analysis of the proteins prepared from ischemic tissue showed significant increase in the 66 and 80 kDa components; where a marked decrease in the 260 kDa protein occurred in the ischemic and para-ischemic tissues. The 66 kDa and 80 kDa proteins stained intensely with anti-serum protein antibodies, indicating that they are related to plasma components. Moreover, the 66 kDa band had the same electrophoretic mobility as bovine serum albumin used as a standard molecular size marker. The 80 kDa band was identified as transferrin by staining with the specific antibody. Transferrin was immunolocalized in the penumbra of cerebral cortex, hippocampal CA1 region and dentate gyrus of the ischemic cerebral hemisphere. The present results suggest that alteration in the brain content of 66 kDa (albumin), 80 kDa (transferrin) and 260 kDa (unidentified) proteins may reflect early effects of focal ischemia.     

Temporal and regional changes of superoxide dismutase and glutathione peroxidase activities in rats exposed to focal cerebral ischemia

Reactive oxygen species are important cause of tissue injury during cerebral ischemia and reperfusion (I/R). Superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) are intracellular enzymes responsible for endogenous antioxidant defense of tissues affected by I/R. The aim of this study was to examine temporal and regional changes of SOD and GSH-Px activities in animals exposed to transient focal cerebral ischemia. Male Wistar Hannover rats were subjected to the right middle cerebral artery occlusion for 2?h. The animals were sacrificed immediately, 0·5, 1, 2, 3, 6, 24, 48, 72 or 168?h after ischemic procedure. SOD and GSH-Px activities were determined spectrophotometrically in the hippocampus and parietal cortex, both unilaterally and contralaterally to the occlusion. Sham-operated animals were used as the control group. Our results indicated that transient focal cerebral ischemia causes significant changes in SOD activities in the hippocampus and parietal cortex such as in GSH-Px activities in the parietal cortex, unilaterally and contralaterally to the lesion in rats during different reperfusion periods. Statistically significant activation of GSH-Px was registered neither in the right nor in the left hippocampus of ischemic animals. Copyright ? 2012 John Wiley & Sons, Ltd.     

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.     

Increased expression of HIF-1alpha, nNOS, and VEGF in the cerebral cortex of anemic rats

This study tested the hypothesis that specific hypoxic molecules, including hypoxia-inducible factor-1alpha (HIF-1alpha), neuronal nitric oxide synthase (nNOS), and vascular endothelial growth factor (VEGF), are upregulated within the cerebral cortex of acutely anemic rats. Isoflurane-anesthetized rats underwent acute hemodilution by exchanging 50% of their blood volume with pentastarch. Following hemodilution, mean arterial pressure and arterial Pa(O(2)) values did not differ between control and anemic rats while the hemoglobin concentration decreased to 57 +/- 2 g/l. In anemic rats, cerebral cortical HIF-1alpha protein levels were increased, relative to controls (1.7 +/- 0.5-fold, P < 0.05). This increase was associated with an increase in mRNA levels for VEGF, erythropoietin, CXCR4, iNOS, and nNOS (P < 0.05 for all), but not endothelial NOS. Cerebral cortical nNOS and VEGF protein levels were increased in anemic rats, relative to controls (2.0 +/- 0.2- and 1.5 +/- 0.4-fold, respectively, P < 0.05 for both). Immunohistochemistry demonstrated increased HIF-1alpha and VEGF staining in perivascular regions of the anemic cerebral cortex and an increase in the number of nNOS-positive cerebral cortical cells (3.2 +/- 1.0-fold, P < 0.001). The nNOS-positive cells costained with the neuronal marker, Neu-N, but not with the astrocytic marker glial fibrillary acidic protein (GFAP). These nNOS-positive neurons frequently sent axonal projections toward cerebral blood vessels. Conversely, VEGF immunostaining colocalized with both neuronal (NeuN) and astrocytic markers (GFAP). In conclusion, acute normotensive, normoxemic hemodilution increased the levels of HIF-1alpha protein and mRNA for HIF-1-responsive molecules. nNOS and VEGF protein levels were also increased within the cerebral cortex of anemic rats at clinically relevant hemoglobin concentrations.     

Effects of Metabotropic Glutamate Receptor Stimulation on Cerebral O2 Consumption and Blood Flow During Focal Cerebral Ischemia in Rats

This investigation was performed to evaluate the effects of ACPD [(1S, 3R)-1-aminocyclopentane-1,3-dicarboxylic acid], a metabotropic glutamate receptor agonist, on cerebral O₂ consumption during focal cerebral ischemia. Male Wistar rats were placed in control (n = 7) and ACPD (n = 7) groups under isoflurane anesthesia. Twenty minutes after middle cerebral artery (MCA) occlusion, gauze sponges with 10^–5 M ACPD or normal saline were placed on the ischemic cortex (IC) for a period of 40 min and were changed every 10 min. One hour after MCA occlusion, regional cerebral blood flow (rCBF) was determined using the C¹⁴-iodoantipyrine autoradiographic technique. Regional arterial and venous oxygen saturation were determined using microspectrophotometry. There were no statistical differences in vital signs, blood gases, and hemoglobin between the groups. In the control group, the cerebral blood flow and oxygen consumption of the IC were significantly lower than the contralateral cortex (rCBF: 45 ± 11 vs. 110 ± 11 ml/min/100 g, O₂ consumption: 2.9 ± 0.4 vs. 5.4 ± 1.1 ml O₂/min/100 g). ACPD did not change regional cerebral blood flow of the IC, but did significantly increase the oxygen extraction (7.8 ± 0.2 vs. 6.9 ± 0.3 ml O₂/100 ml) and oxygen consumption of the IC (4.3 ± 1.5 vs. 2.9 ± 0.4) compared to the control IC. Our data demonstrated that topical application of 10^–25 M ACPD to the ischemic area worsened cerebral O₂ balance. These data suggest that metabotropic glutamate receptors are not maximally activated during ischemia in the temporal cortex.