Diaspirin Cross-linked Hemoglobin Research Articles

Effect of subarachnoid administration of alpha-alpha diaspirin crosslinked hemoglobin on cerebral blood flow in rats.

As extravasated red blood cells have been implicated in the pathogenesis of perfusion deficits after subarachnoid hemorrhage, alpha-alpha diaspirin crosslinked hemoglobin (DCLHb) might have a detrimental effect on cerebral perfusion after subarachnoid hemorrhage. We evaluated the effect of subarachnoid administration of DCLHb on cerebral blood flow (CBF). Rats were randomized to receive one of the following solutions into the cisterna magna: Control-0.3 ml of mock cerebrospinal fluid; Blood-0.3 ml of autologous blood; DCLHb-0.3 ml of 10% DCLHb. After 20-min, the area of cerebral hypoperfusion was determined (CBF < 40 ml.100g-1.min-1). The area of hypoperfusion (% area of a coronal brain section, mean +/- SD) was greater in the Blood group (58 +/- 16) than the DCLHb (16 +/- 7) and Control (5 +/- 5) groups (p < 0.05), and was greater in the DCLHb group than the Control group (p < 0.05). These data support a hypothesis that extravasation of blood from the intravascular to the subarachnoid space induces cerebral hypoperfusion. Moreover, the data support the hypothesis that although extravasated molecular hemoglobin decreases CBF, the adverse effect is not as severe as a similar volume of blood.

Hypervolemic-Hemodilution During Cerebral Ischemia in Rats

In a rat model of middle cerebral artery occlusion (MCAo) and reperfusion (120 min), previous studies have demonstrated that hemodilution with molecular hemoglobin decreases ischemic brain injury. However, long-term recovery data on the therapeutic efficacy of molecular hemoglobin for cerebral ischemia are lacking. Accordingly, we assessed the effect of hemodilution, with alpha-alpha diaspirin cross-linked hemoglobin (DCLHb, 10 g/dl) on neurologic outcome and infarct volume after 120 min of MCAo and 72 h of reperfusion. Ischemia was achieved by passing a 0.26-mm suture, via the external carotid artery, to internally occlude the middle cerebral artery. Immediately after MCAo, the rats were randomized to one of the following groups: Control-hematocrit not manipulated (44%); 30/Hct-hematocrit maintained at 30% with DCLHb; or 16/Hct-hematocrit maintained at 16% with DCLHb. After 120 min of MCAo, the suture was removed and the rats allowed to recover. Daily neurologic examinations were performed, and after 72 h, the brains were analyzed for infarct volume with TTC stain. Infarct volume (mm3) was less in the 30/Hct group (67 +/- 10; mean +/- SD) than in the Control group (141 +/- 17); and less in the 16/Hct group (40 +/- 12) than the other two groups (p < 0.05). Neurologic outcome was improved in both hemodilution groups versus the Control group (p < 0.05). These data are consistent with previous studies, performed in a model of short-term reperfusion, which indicate a dose-dependent decrease in ischemic injury by DCLHb.

Mechanism of the contractile effect of diaspirin-cross-linked hemoglobin in rat isolated aorta strip denuded of endothelium as revealed using an oil-immersion procedure

Diaspirin-cross-linked hemoglobin (DCLHb) is a chemically modified hemoglobin (Hb) (i.e., alpha-subunits are cross-linked by a covalent bond) currently being tested as a potential oxygen-carrying blood substitute. It was examined for possible vasoactive properties, using the rat isolated aorta strip denuded of endothelium. In this experimental model, DCLHb (1.6-155 microM) was found to be inactive as a vasoconstrictor when added to the Krebs medium but to elicit contractile responses once the Krebs medium containing DCLHb was replaced by mineral oil, a procedure that favors the sequestration of a fixed amount of DCLHb within a substantially reduced volume of extracellular fluid. The contractile activity of DCLHb in our experimental model (i.e., prior exposure of tissues to drugs in the Krebs medium followed by replacement of the Krebs medium by mineral oil) was mimicked by methemoglobin and metmyoglobin, but not by cytochrome c, albumin, hemin, hematin, Fe2+, and a variety of hemorphins. It was abolished by indomethacin, SQ-29548 (prostaglandin H2-thromboxane A2 receptor antagonist), thiourea, or N-2-mercaptopropionylglycine (MCPG), reduced partially by verapamil, but not affected by dazmegrel, MK-886 (leukotriene biosynthesis inhibitor), dimethylsulfoxide, vitamin C or E, deferoxamine, NG-nitro-L-arginine, naloxone, and a variety of other drug receptor antagonists (e.g., prazosin) and protease inhibitors (e.g., pepstatin). Rat aorta strips denuded of endothelium exhibited contractile responses to arachidonic acid added in the Krebs medium (i.e., with no mineral oil added afterwards). Such contractile activity was reduced by SQ-29548, thiourea, or MCPG. Addition of U-46619 (prostaglandin H2-thromboxane A2 mimetic) to the Krebs medium also elicited contractile responses in rat aorta strips denuded of endothelium. Such contractile activity was reduced by SQ-29548, thiourea, or verapamil but not by MCPG. Within the limitations of our experimental approach, these results suggest that (1) the contractile activity of DCLHb in rat aorta strips denuded of endothelium following replacement of the Krebs medium by mineral oil involves the participation of a secondary mediator, which could be a vasoconstrictor metabolite of arachidonic acid; (2) the participation of reactive oxygen species, potential degradation products of DCLHb (e.g., heme, Fe2+, hemorphins), or other mediators in the contractile activity of DCLHb is unlikely; and (3) Ca2+ entry into target cells might be involved in the process by which DCLHb elicits its contractile activity in our experimental model.

Combined neuroprotection and reperfusion therapy for stroke. Effect of lubeluzole and diaspirin cross-linked hemoglobin in experimental focal ischemia.

In search of a better treatment for acute ischemic stroke, we evaluated the use of lubeluzole and hemodilution with diaspirin cross-linked hemoglobin (DCLHb) therapy to test whether treatment with two complementary acting compounds provides more potent protection than either treatment alone. We used unilateral reversible middle cerebral artery (MCA) and common carotid artery (CCA) occlusion of various durations in Long-Evans rats to produce ischemic cortical lesions. We calculated the average maximal lesion volume (Volmax) and the time required to produce half maximal lesion size (T50) in control animals (n = 31) and evaluated the effects on cerebral perfusion and infarct size of treatment with lubeluzole (n = 23), hemodilution (to 30% hematocrit) with albumin (n = 17) or DCLHb (n = 23), and combined lubeluzole + DCLHb therapy initiated 15 minutes after MCA/CCA occlusion. The Volmax produced by MCA/CCA occlusion in control animals was 138.5 +/- 7.7 mm3, and T50 was 98.5 +/- 10.2 minutes. Lubeluzole alone reduced Volmax by 53% with no significant effect on T50. In contrast to lubeluzole, DCLHb hemodilution prolonged T50 by 68% with no significant effect on Volmax. Prolongation of T50 by DCLHb was not due to hemodilution itself, since a similar degree of hemodilution with albumin had no effect. Finally, combined lubeluzole+DCLHb rescued 72% of the tissue and augmented the effect of lubeluzole alone by 40% (Volmax, 66.3 +/- 13.0 versus 39.4 +/- 12.2 mm3) while prolonging T50 by 31%. Combination therapy for acute stroke using compounds with complementary action can result in more complete attenuation of neuronal damage and demonstrates the possible clinical utility of combined neuroprotective and reperfusion therapies.

Effect of oncotic pressure of diaspirin cross-linked hemoglobin (DCLHb) on brain injury after temporary focal cerebral ischemia in rats.

Previous studies have shown that diaspirin cross-linked hemoglobin (DCLHb, 10 g/dL) decreases cerebral ischemia and the resultant injury in a dose-dependent manner, requiring large volumes of DCLHb for maximum efficacy. We assessed the effect of a more concentrated (20 g/dL) and more hyperoncotic preparation of DCLHb on cerebral infarction volume. Immediately after middle cerebral artery occlusion, rats were randomized to one of the following groups: Control, hematocrit not manipulated; 10/Hb, hematocrit decreased to 30% with 10% DCLHb (oncotic pressure 43 mm Hg); 7.5/Alb, hematocrit decreased to 30% with 7.5% albumin (oncotic pressure 43 mm Hg); 20/Hb, the same dose of DCLHb (20%, oncotic pressure 129 mm Hg) as the 10/HB group (half the volume); or 15/Alb, the same dose of albumin (15%, oncotic pressure 130 mm Hg) as the 7.5/Alb group half the volume). After 90 min of ischemia, 72 h of reperfusion was allowed. Infarction volume (mm3, mean +/- sd) was less in the DCLHb groups (10/Hb = 79 +/- 17; 20/HB = 51 +/- 14) than the oncotically matched albumin groups (7.5/Alb = 124 +/- 21; 15/Alb = 85 +/- 18) and the Control group (135 +/- 17) (P < 0.05). These data indicate that in this model of cerebral ischemia, DCLHb decreases ischemic brain injury more effectively than albumin, and that a hyperoncotic preparation of DCLHb is preferable.

Effect of Oncotic Pressure of Diaspirin Cross-Linked Hemoglobin (DCLHb Trademark) on Brain Injury After Temporary Focal Cerebral Ischemia in Rats

Effect of Oncotic Pressure of Diaspirin Cross-Linked Hemoglobin (DCLHb Trademark) on Brain Injury After Temporary Focal Cerebral Ischemia in Rats

Diaspirin Cross-Linked Hemoglobin Is Efficacious in Gut Resuscitation as Measured by a GI Tract Optode

The objective of this study was to compare the efficacy of diaspirin cross-linked hemoglobin (DCLHb) with that of standard resuscitative fluids in restoring intestinal mucosal oxygenation and villous architecture after hemorrhage. Male rats were bled to a base deficit of 5 +/- 2 nmol/l under propofol anesthesia and monitored for 90 minutes postresuscitation with DCLHb, blood, lactated Ringer's solution, albumin, or nothing (DNR) for mucosal oxygen tension (Pmo2) and physiologic and laboratory parameters. Small intestinal histologic specimens were obtained and scored independently by two investigators blinded to therapy on a scale of 0 (normal) to 4 (worst). All treatments restored Pmo2; only DCLHb did so without exceeding baseline values. For untreated rats (DNR), Pmo2 was not restored. Normal mucosal architecture was maintained only in DCLHb-treated rats. As Pmo2 increased, mucosal score improved. In a rat model of controlled hemorrhage, Pmo2 changes measured by an optode correlated with gut histological abnormalities. By these criteria, DCLHb is superior to crystalloid, colloid, and blood in gut resuscitation.

THE EFFECTS OF DIASPIRIN CROSS-LINKED HEMOGLOBIN IN SEPSIS

We tested the hypothesis that diaspirin cross-linked hemoglobin (DCLHb; Baxter Healthcare Corp.) would improve blood pressure, organ perfusion, and mortality during sepsis. Rats were catheterized to assess general hemodynamics (protocol 1) or regional blood flow (protocol 2). Sepsis was induced by intraperitoneal introduction of a cecal slurry (100 mg/kg). In protocol 1, rats received either 100 or 250 mg/kg DCLHb, or albumin at 1, 2, or 4 h after sepsis induction. Hemodynamics were recorded at these times and daily for 72 h. DCLHb increased blood pressure, prevented 72 h leukocytosis, and reduced mortality, but the timing of DCLHb administration was crucial. In protocol 2 only moribund septic animals received 100 mg/kg DCLHb or iso-oncotic albumin i.v. Hemodynamics and regional organ blood flows were measured at baseline, immediately before and after treatment, and at 24 h. DCLHb immediately increased blood pressure with no changes in cardiac output, heart rate, or regional perfusion. DCLHb increased regional perfusion to vital areas at 24 h (compared to albumin group). Distribution of cardiac output in albumin-treated rats was significantly skewed toward skeletal muscle at a time when cardiac output was significantly lower as compared with DCLHb treated animals. In conclusion, DCLHb safely elicited a pressor response, and improved regional perfusion to selected tissues. However, DCLHb benefits were best obtained when given within a specific time frame.

Early Resuscitation with Low-Volume PolyDCLHb Is Effective in the Treatment of Shock Induced by Penetrating Vascular Injury

To study the efficacy of an oxygen-carrying solution in early resuscitation of hemorrhagic shock induced by penetrating vascular injury. Experimental study with anesthetized rats. Severe hemorrhagic shock was induced by a 25-gauge needle puncture to the infrarenal aorta. Forty animals were resuscitated 10 minutes after injury with either lactated Ringer's solution (LR; 60 mL/kg), 7.5% hypertonic saline (HTS; 5 mL/kg), or modified diaspirin cross-linked hemoglobin (PolyDCLHb; 5 or 20 mL/kg) or were not resuscitated (NR) and followed for 6 hours. Total blood loss was similar in all treatment groups. Mean arterial pressure was restored to baseline values, base deficit was corrected to base excess, and venous oxygen saturation improved with PolyDCLHb and more slowly with LR but persisted below baseline values with HTS and NR. The 6-hour mortality rates were zero of eight (low-dose PolyDCLHb), three of eight (high-dose PolyDCLHb), two of eight (LR), six of eight (HTS), and six of eight (NR). Early resuscitation with low-volume hemoglobin is effective in restoring tissue perfusion and improving survival in uncontrolled hemorrhagic shock.

Resuscitation with increasing doses of diaspirin crosslinked hemoglobin in swine.

This study examined the effects of administering 0.5, 4, 10, and 30 mL/kg of Diaspirin Crosslinked Hemoglobin (DCLHb) in a swine model of non-lethal hemorrhagic shock. Thirty unanesthetized animals were bled (30 mL/kg, 1 mL/kg/min) and either recovered without treatment (Untreated Control, UC) or infused with 10 g/dL DCLHb (0.5, 4.0, 10 or 30 mL/kg at 1 mL/kg/min) or Lactated Ringer (LR, 90 mL/kg at 3 mL/kg/min). DCLHb caused dose-related increases in MAP. Both the 10 and 30 mL/kg doses of DCLHb increased MAP more than UC or LR. Lower doses of DCLHb and LR had effects on MAP similar to UC. After hemorrhage, CO increased in all groups. The effect of DCLHb on CO was dose-related. Only LR and 30 mL/kg of DCLHb transiently (through 90 min) increased CO more than UC. CO in animals given lower doses of DCLHb was comparable to UC. DCLHb (10 and 30 mL/kg) improved base excess and lactate concentrations, two indices of global perfusion, more rapidly and to a greater extent than either UC or LR. In this swine model of hemorrhage, even small doses of DCLHb exerted measurable beneficial effects on blood pressure and perfusion.

Use of diaspirin cross-linked hemoglobin in critically ill patients

Use of diaspirin cross-linked hemoglobin in critically ill patients

Pharmacologic profile of diaspirin cross-linked hemoglobin in hemodialysis patients

Various hemoglobin compounds have been evaluated as potential oxygen-carrying, blood volume expanders, but toxicity has prevented clinical application. Diaspirin cross-linked hemoglobin (DCLHb) represents a modified hemoglobin compound that is derived from human red blood cells and maintained in a tetrameric configuration by cross-linkages between the two alpha chains of the hemoglobin molecule. In a randomized, placebo-controlled, single-blind, cross-over trial, DCLHb's safety and pharmacologic parameters were evaluated in 18 subjects receiving chronic hemodialytic therapy. A 30-minute infusion of 25, 50, or 100 mg/kg DCLHb or placebo was given at the start of routine hemodialysis. One week later, the alternate treatment (placebo or DCLHb) was administered. Maximum plasma hemoglobin concentrations and terminal half-life values were calculated for each dosage group. Dialysate was collected and assayed for hemoglobin. Changes in systolic and diastolic blood pressure from baseline and the volume of hypertonic saline administered for treatment of hypotension during hemodialysis were measured. The maximum plasma hemoglobin concentrations increased with DCLHb dose and occurred at the end of DCLHb infusion. The mean (±SD) terminal half-life ranged from 2.1 ± 1.0 hours in the 25 mg/kg DCLHb group to 4.3 ± 1.4 hours in the 100 mg/kg group, but did not differ significantly between groups. Mean baseline plasma hemoglobin connected areas under the plasma concentration-time curves increased from 89 to 1,136 mg/hr/dL across the fourfold dose range. Diaspirin cross-linked hemoglobin was not dialyzable as none was detected in dialysate. The maximum increase in systolic blood pressure from baseline increased significantly with DCLHb dose compared with placebo ( P < 0.05). During hemodialysis with DCLHb infusions, significantly less hypertonic saline was administered compared with hemodialysis with placebo infusions, implying greater blood pressure stability with DCLHb. No significant adverse events were noted. Further efficacy and safety studies with multiple-dose DCLHb administration are warranted in hemodialysis patients.

Diaspirin cross-linked hemoglobin does not increase brain oxygen consumption during hypothermic cardiopulmonary bypass in rabbits.

Decreased erythrocyte deformability due to cardiopulmonary bypass (CPB) and/or hypothermia, may result in brain capillary beds that have decreased erythrocyte transit, resulting in a generalized impairment of brain oxygenation during CPB. Because brain capillary plasma flow continues even when erythrocyte flow is absent, the authors' hypothesized augmentation of plasma oxygen content with a non-erythrocyte-associated oxygen transport molecule would increase brain oxygen uptake during hypothermic CPB. Anesthetized New Zealand white rabbits, maintained on CPB at 27 degrees C, were randomized to one of three groups. In group 1 (n = 13), plasma oxygen content was increased by administration of alpha-alpha diaspirin cross-linked hemoglobin. In this group, pretreatment with 0.5 mg/kg verapamil was necessary to prevent hypertension. In group 2 (n = 13), alpha-alpha diaspirin cross-linked hemoglobin was not administered, but verapamil was given as before (control). In group 3 (n = 13), neither alpha-alpha diaspirin cross-linked hemoglobin nor verapamil was administered (control). At 60 min of CPB, cerebral blood flow (microspheres) and cerebral metabolic rate for oxygen (Fick) were determined. Systemic physiologic variables did not differ among groups. Although total arterial oxygen content was equivalent in all groups (approximately 12.1 ml O2/dl), the alpha-alpha diaspirin cross-linked hemoglobin group had a much greater proportion of the total arterial oxygen content present in a non-erythrocyte-associated form, 29 +/- 5% versus 6 +/- 2% and 5 +/- 3%, in groups 2 and 3, respectively. Nevertheless, neither cerebral blood flow (approximately 34 ml.100g-1.min-1) nor cerebral metabolic rate for oxygen (approximately 1.2 ml O2.100g-1.min-1) differed among groups. Because oxygen was equally available to the brain in all groups, independent of whether oxygen was associated with erythrocytes or not, it was concluded that erythrocyte/capillary interactions do not limit oxygen transfer from blood to brain during moderately hypothermic CPB. The hypertensive response to alpha-alpha diaspirin cross-linked hemoglobin during CPB is probably a result of nitric oxide scavenging.

Role of NO mechanism in cardiovascular effects of diaspirin cross-linked hemoglobin in anesthetized rats.

The role of nitric oxide (NO) in the cardiovascular actions of diaspirin cross-linked hemoglobin (DCLHb) was studied in anesthetized rats. The regional circulatory and systemic hemodynamic effects of DCLHb (400 mg/kg iv) were studied using a radioactive microsphere technique in control (untreated) and L-arginine (a NO precursor) pretreated rats. DCLHb produced a significant increase in blood pressure (75%), cardiac output (42%), stroke volume (36%), and total peripheral resistance (45%), without affecting heart rate, when administered to control rats. L-Arginine pretreatment significantly attenuated DCLHb-induced systemic hemodynamic effects. DCLHb-induced increase in blood flow to the skin and spleen was completely blocked, and that to the heart was partially blocked, by L-arginine pretreatment, suggesting that cardiovascular actions induced by DCLHb could be antagonized by the NO precursor L-arginine. The NO synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME) produced significant increases in regional vascular resistance, leading to a decrease in blood flow to all the organs except the heart, where an increase in blood flow and a decrease in vascular resistance was observed. DCLHb, when administered in L-NAME-pretreated rats, accentuated the decrease in blood flow to the gastrointestinal system, spleen, mesentery and pancreas, skin, and musculoskeletal system. These studies provide evidence that the NO precursor L-arginine can attenuate the effects of DCLHb and that DCLHb can potentiate the effect of the NOS inhibitor L-NAME. The role of NO in the mechanism of action of DCLHb was further studied by estimating plasma guanosine 3',5'-cyclic monophosphate (cGMP) in control, DCLHb-treated, L-NAME-treated, and L-NAME followed by DCLHb-treated rats. DCLHb and L-NAME significantly decreased the concentration of circulating cGMP in blood plasma. L-NAME pretreatment potentiated DCLHb-induced decrease in cGMP levels. Because the formation of cGMP is stimulated by NO, these studies provide additional evidence for the involvement of NO in the mechanism of action of DCLHb. It is concluded that NO plays an important role in the cardiovascular effects of DCLHb.

Effects of polymerization on the oxygen carrying and redox properties of diaspirin cross-linked hemoglobin

Human hemoglobin site specifically cross-linked with bis(3,5-dibromosalicyl)fumarate results in a low oxygen affinity hemoglobin-based red cell substitute (α-DBBF). Polymerization of α-DBBF by bis(maleoylglycylamide) polyethylene glycol (BMAA-PEG) yields poly α-DBBF which offers the added benefits of reduced renal clearance and increased retention in the vascular circulation. Oxygen equilibrium curves for poly α-DBBF are slightly left-shifted (higher O 2 affinity) compared to those of α-DBBF; with a diminished cooperativity and a reduced Bohr effect. In rapid mixing experiments (oxygen dissociation and carbon monoxide binding), poly α-DBBF exhibits a several fold increase in the overall rate of deoxygenation and carbon monoxide binding kinetics over its cross-linked counterpart. The rate of nitric oxide binding to the oxidized form of poly α-DBBF shows little or no change compared to the intramolecularly cross-linked derivative. The reduction of cyanomet poly α-DBBF by dithionite is several fold faster than that of HbA 0 and α-DBBF whereas the slow subsequent cyanide dissociation from the ferrous iron remained unchanged among all proteins. The propensity of poly α-DBBF for auto-oxidation is slightly enhanced over α-DBBF whereas the extent of oxidative modification by hydrogen peroxide is very similar. Polymerization appears to selectively modify ligand interactions and redox kinetics of the tetrameric cross-linked form which reflects a possibly more open heme pocket. The data suggests that changes in oxygenation properties of hemoglobin brought about by a given modification are not necessarily predictive of other functional changes.

Diaspirin crosslinked hemoglobin (DCLHb) does not affect the anesthetic potency of isoflurane in rats.

Hemoglobin solutions are being developed as oxygen carrying fluids for multiple clinical indications. Despite an early report of accentuation of ether anesthesia, the effect of hemoglobin on anesthetic potency has not been assessed. We assessed the effect of alpha-alpha diaspirin crosslinked hemoglobin (DCLHb) on the anesthetic requirement of isoflurane necessary to keep rats unresponsive to noxious stimuli (1.0 MAC [minimum alveolar concentration]). During isoflurane administration, each rat received one of the following fluid regimens: 44Hct/N-normal hematocrit and volume; 44Hct/H-8.0 ml of donor blood given as a hypervolemic bolus; 30Hct/H-5.0 ml of DCLHb given as an exchange transfusion and 8.0 ml as a hypervolemic bolus; or 16Hct/H-15.0 ml of DCLHb given as an exchange transfusion and 8.0 ml as a hypervolemic bolus. MAC was determined using a standard tail clamp technique. The isoflurane requirement to achieve 1.0 MAC was not different between the four groups. These results are consistent with a hypothesis that DCLHb does not change the anesthetic state.

Effects of Halogenated and Non-Halogenated Anesthetics on Diaspirin Cross-Linked Hemoglobin™-Induced Contractions of Porcine Pulmonary Veins

Diaspirin crosslinked hemoglobin (DCLHb) is a resuscitative fluid presently undergoing clinical trials. Administration of DCLHb is associated with an elevation of mean arterial pressure in vivo and contraction of isolated blood vessels in vitro. The mechanisms for the vascular actions are unknown but may be due to inhibition of nitric oxide (NO). Halothane has been reported to inhibit NO induced relaxation. We examined the effect of anesthetics on DCLHb induced contraction of blood vessels. Porcine pulmonary veins were excised, cut into rings and placed in organ chambers filled with 25 ml Krebs-Ringer solution (37 degrees C). Following equilibration at their optimal length the rings were exposed to increasing concentrations of serotonin(10(-8)M-10(-5)M). Endothelial activity was confirmed by relaxation greater than 80% with ACh (10(-6)M). DCLHb (1.5 x 10(-8)M to 6 x 10(-7)M) contracted porcine pulmonary veins (1.04 +/- 0.17g to 3.45 +/- 0.22g), and halothane (0.5% and 1%) significantly inhibited these DCLHb induced contractions in a dose-related manner (-41.6 +/- 8.1% and -73.3 +/- 8.2%, respectively). At equi-molar concentrations, isoflurane had no inhibitory activity. The relative effect of these volatile anesthetics is consistent with their inhibitory actions on other heme containing proteins. Propofol (10(-5)M) only has inhibitory effects on lower concentrations of DCLHb. Fentanyl did not have inhibitory effects. These results suggest that halogenated anesthetics may interact with the heme iron of DCLHb and inhibit its binding with NO.

Diaspirin cross-linked hemoglobin improves neurological outcome following reversible but not irreversible CNS ischemia in rabbits.

Hemodilution using modified hemoglobin solutions may reduce ischemic central nervous system injury. Purified diaspirin cross-linked hemoglobin (DCLHb) is a cell-free hemoglobin that is intramolecularly cross-linked between the two alpha subunits, resulting in enhanced oxygen offloading to tissues and increased half-life. In the present experiments, we evaluated the ability of DCLHb to reduce neurological damage in two rabbit stroke models. In a reversible spinal cord ischemia model, ischemia of the caudal lumbar spinal cord was produced by temporary occlusion of the abdominal aorta. In an irreversible model of cerebral ischemia, plastic microspheres (50 microns) were injected into the internal carotid artery and lodged in the cerebral microvasculature. DCLHb was administered 5 minutes after initiation of ischemia as either a 10-mL/kg infusion, 10-mL/kg exchange transfusion, or a 20-mL/kg infusion. Control animals received human serum albumin that was oncotically matched to the DCLHb. In the spinal cord model, DCLHb significantly increased the duration of ischemia required to produce permanent paralysis from 27.33 +/- 8.71 minutes (mean +/- SD) in controls to 42.59 +/- 10.10 minutes in the 10-mL/kg exchange transfusion group and to 40.82 +/- 18.16 minutes in the 20-mL/kg infusion condition (P < .05). DCLHb did not significantly reduce neurological damage in the microsphere embolization model. These data suggest that cross-linked hemoglobin reduces neurological damage after reversible central nervous system ischemia and that this is not attributable to hemodilution or hypervolemia only.

THE EFFICACY OF DIASPIRIN CROSSLINKED HEMOGLOBIN SOLUTION RESUSCITATION IN A MODEL OF UNCONTROLLED HEMORRHAGE

Controversy exists whether early aggressive fluid therapy in the setting of uncontrolled hemorrhage worsens outcome by increasing blood loss from injured vessels. Since diaspirin crosslinked hemoglobin (DCLHb) is a vasoactive, oxygen-carrying solution, we compared the effects of DCLHb with other resuscitative fluids on blood loss, hemodynamics, and tissue oxygen delivery in a model of uncontrolled hemorrhage. Anesthetized rats (250-350 g) were subjected to a 50% tail transection and resuscitated 15 minutes later with 1:1 DCLHb, 3:1 lactated Ringer's solution (LR), 1:1 hypertonic saline (7.5% HTS), or 1:1 human serum albumin (8.3% HSA) based on initial volume of blood loss (average 4.7 +/- 0.3 mL/kg). An unresuscitated group served as a control. Cumulative blood loss was measured at 5 hours postresuscitation. By 15 minutes after tail transection, mean arterial pressure (MAP) decreased 19.2 +/- 3.8 mm Hg from the baseline value (102 +/- 5 mm Hg). The DCLHb solution restored and maintained MAP and subcutaneous tissue oxygen tension at baseline values better than all other resuscitative fluids. Although blood loss in DCLHb-treated animals was greater than in unresuscitated animals, it was no different from other resuscitative fluids and less than with HSA. There was no difference in 24-hour survival between all treatment groups. In conclusion, DCLHb elevates MAP but does not exacerbate blood loss or compromise tissue oxygen delivery compared with other resuscitative fluids in this model of uncontrolled hemorrhage.

Effect of stroma-free hemoglobin and diaspirin cross-linked hemoglogbin on the regional circulation and systematic hemodynamics

The effects of unmodified stroma-free hemoglobin (SFHb) and diaspirin cross-linked hemoglobin (DCLHb) on the regional blood circulation and systematic hemodynamics were studied in rats using a radioactive microsphere technique. SFHb and DCLHb increased mean arterial blood pressure without affecting heart rate. SFHb produced a 24.9 % decrease in the cardiac output while DCLHb produced an 44.8 % increase in the cardiac output. Stroke volume was decreased (−27.3 %) by SFHb and increased (+36.4 %) by DCLHb. Total peripheral resistance increased with both SFHb and DCLHb. DCLHb increased blood flow to the heart, spleen, stomach, small intestine and skin, and had no effect on blood flow to the brain, kidneys, liver, mesentery, pancreas, caecum, large intestine and musculo-skeletal system. In contrast, in animals infused with SFHb, blood flow decreased to the kidneys, liver and spleen, increased to the heart, small intestine and skin, and had no effect to the brain, caecum, large intestine and musculo-skeletal system. DCLHb had no effect on vascular resistance in any organ except for an increase in the musculo-skeletal system. In contrast, SFHb increased vascular resistance in the kidneys, liver, spleen, skin, mesentery and pancreas, and had no effect on vascular resistance in the musculo-skeletal system, brain, heart, stomach, small intestine, caecum and large intestine. SFHb had no effect on distribution of cardiac output to the brain, gastrointestinal tract (GIT), kidneys, skin, musculo-skeletal and portal system, while DCLHb significantly decreased the percent cardiac output to the musculo-skeletal system. DCLHb did not affect the distribution of cardiac output to the brain, GIT, kidneys, skin and portal system. SFHb and DCLHb increased the percent cardiac output to the heart. It is concluded that similar concentrations and doses of DCLHb and SFHb produce different effects on the regional blood circulation and systematic hemodynamics.