Diaspirin Cross-linked Hemoglobin Research Articles

Subarachnoid molecular hemoglobin after subarachnoid hemorrhage in rats: effect on the area of hypoperfusion.

A previous study indicated that diaspirin-crosslinked hemoglobin (DCLHb) decreases cerebral ischemia after subarachnoid hemorrhage. However, the study was limited in that DCLHb was given to animals with an intact vasculature. As extravasated hemoglobin has been implicated in the pathogenesis of cerebral vasospasm, DCLHb in the subarachnoid space might in theory have a detrimental effect on cerebral perfusion after subarachnoid hemorrhage. In the current study, autologous blood was administered into the cistema magna of isoflurane-anesthetized rats. After 30 minutes, the animals received one of the following in the cistema magna (n=8 for each group): The control group received mock cerebral spinal fluid, the "blood" group received autologous blood, the "DCLHb" group received DCLHb, and the "Alb" group received human serum albumin. After 20 minutes, areas of reduced cerebral blood flow (CBF, 0-20 and 21-40 ml/100 g/min) were assessed in five coronal brain sections with 14C-iodoantipyrine. The data were evaluated by analysis of variance. The areas of 0-20 ml/100 g/min CBF were greater in all five brain sections in the Blood group than in the other three groups; were greater in four brain sections in the DCLHb group than in the Control group; and were greater in three brain sections in the Alb group than in the Control group (p < 0.05). The areas of 21-40 ml/100 g/min CBF were greater in three sections in the Blood group than in the other three groups; and were greater in two brain sections in the DCLHb group than in the Alb group (p < 0.05). These data support a hypothesis that subarachnoid blood induces cerebral hypoperfusion, and that although molecular hemoglobin decreases CBF, the potential adverse effects are less than those produced by blood.

Effects of diaspirin cross-linked haemoglobin on post-traumatic cerebral perfusion pressure and blood flow in a rodent model of diffuse brain injury

Diaspirin cross-linked haemoglobin (DCLHb) is a new oxygen carrying blood substitute with vasoactive properties. Vasoactive properties may be mediated via high affinity binding of nitric oxide by the haem moiety. Using a rodent model of head injury combined with ischaemia, we studied the effects of DCLHb on cerebral blood flow (CBF) and intracranial pressure (ICP). Twenty anaesthetized rats were allocated randomly to receive treatment with DCLHb 400 mg kg-1 i.v. or placebo (oncotically matched plasma protein substitute 4.5% i.v.). To produce diffusely increased ICP, after a severe weight drop injury, all animals underwent a 30-min period of bilateral carotid ligation combined with a period of induced hypotension. After reperfusion, DCLHb or placebo was infused and the animals instrumented for measurement of intraventricular ICP and CBF in the region of the sensorimotor cortex using the hydrogen clearance technique. Mean arterial pressure (MAP), ICP, cerebral perfusion pressure (CPP) (CPP = MAP - ICP) and CBF were measured 4 h after injury in all animals. DCLHb significantly reduced ICP from mean 13 (SEM 2) to 3 (1) mm Hg (P < 0.001), increased CPP from 52 (8) to 95 (6) mm Hg (P < 0.001) and increased CBF from 21 (2) to 29 (2) ml 100 g-1 min-1 (P = 0.032). We conclude that DCLHb improved CPP without a reduction in CBF in a rodent model of post-traumatic brain swelling.

Mechanism of the acute pressor effect and bradycardia elicited by diaspirin crosslinked hemoglobin in anesthetized rats

Diaspirin crosslinked hemoglobin (DCLHb) is a chemically stabilizedhemoglobin (Hb) that induces an increase in blood pressure and a decrease of heart rate wheninjected intravenously in some animals. The mechanism by which DCLHb elicits thesehemodynamic effects was studied in pentobarbital-anesthetized, vagotomized rats using a varietyof drugs known for their inhibitory action towards endogenous hemodynamically active systems.The hypertensive episode elicited by DCLHb (100 or 400 mg·kg–1) was attenuatedin animals pretreated with NG-nitro-L-arginine (inhibitor of nitric oxidesynthases) throughout the 30-min period of observation, but it was not reduced in thosepretreated with a variety of sympatholytic drugs (e.g., prazosin), atropine, BIBP-3226(neuropeptide Y antagonist), indomethacin,[1-(Beta-mercapto-Beta,Beta-cyclopentanemethylene propionic acid), 2-(0-methyl)tyrosine]-Arg8 vasopressin (vasopressin antagonist), losartan (angiotensin antagonist),bosentan (endothelin antagonist), or L-arginine- (nitric oxide precursor), compared withcontrol animals. With the exception of propranolol and BIBP-3226, none of the aforenamedinhibitors reduced the amplitude of the bradycardia associated with the pressor effect of DCLHb.These results suggest that: (i) the acute (&lt;30 min) pressor activity of DCLHb inour animal model requires the presence of an endogenous nitric oxide generating system to beexpressed; (ii) the bradycardia elicited by DCLHb might involve the participation ofneuropeptide Y and (or) its NPY-1 receptors, but it is unlikely to involve abaroreceptor-mediated vagal reflex, at least in our animal model.Key words: hemoglobin, nitric oxide, blood pressure, heart rate,DCLHb.

Modulation of resuscitative effect of diaspirin cross-linked hemoglobin by L-NAME in rats.

Diaspirin Cross-linked Hemoglobin (DCLHb), a hemoglobin-based oxygen carrier, improves regional blood circulation and systemic hemodynamics in normal and hemorrhaged rats. The action of DCLHb is partly mediated by its scavenging effect on nitric oxide. This study was undertaken to determine the effect of DCLHb on nitric oxide mechanism in hemorrhagic conditions. We studied the modulation of cardiovascular effects of DCLHb by a nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) in hemorrhaged rats. The base deficit, survival time, oxygen consumption, and blood circulation to the brain, heart, gastrointestinal tract, and kidneys were determined in 1) DCLHb (100 mg/kg, intravenously (i.v.), 2) L-NAME (2 mg/kg, i.v.), 3) L-NAME (2 mg/kg, i.v.) + DCLHb (100 mg/kg, i.v.), and 4) L-arginine (100 mg/kg/h, i.v.) + DCLHb (100 mg/kg, i.v.) treated rats. Hemorrhage was induced in urethane-anesthetized male rats by bleeding them at a rate of approximately .5 to 1 mL/min, until a mean arterial pressure of 35-40 mmHg was achieved. This blood pressure was maintained for 30 min. Sham-operated nonhemorrhaged rats survived for >300 min, whereas hemorrhaged rats survived for only 85+/-31 min. Hemorrhage significantly increased base deficit and decreased oxygen consumption. A significant decrease in heart rate, mean arterial pressure, cardiac output, stroke volume, and in blood flow to the gastrointestinal tract and kidneys was observed after hemorrhage. Resuscitation with DCLHb produced a significant increase in survival time, oxygen consumption, heart rate, mean arterial pressure, cardiac output, total peripheral resistance, and blood flow to the brain, heart, and kidneys. In contrast, resuscitation with L-NAME did not improve base deficit, survival time, oxygen consumption, systemic hemodynamics, or regional blood flow. L-arginine pretreatment did not affect DCLHb-induced resuscitation of hemorrhaged rats. Furthermore, L-NAME (pretreated or co-administered) attenuated the resuscitative effect of DCLHb. These data suggest that nitric oxide mechanism may not be the only mechanism involved in the resuscitative effect of DCLHb.

Absence of Immunogenicity of Diaspirin Cross-Linked Hemoglobin in Humans

Diaspirin cross-linked hemoglobin (DCLHb) is an intramolecularly cross-linked hemoglobin-based oxygen carrier being developed as a therapy for acute blood loss. We report here the absence of immunogenicity of DCLHb in patients enrolled in phase II and III clinical trials of DCLHb. Two very sensitive immunoassays, an enzyme-linked immunosorbent assay (ELISA) and a Western blot assay, were developed and validated for this assessment. The DCLHb-antibodies used in these assays were raised in monkeys, had similar affinities for DCLHb and native human hemoglobin (SFHb), and showed cross-reactivity for subunits of DCLHb and SFHb on the Western blot, suggesting that these antibodies were elicited as a xenogenic response to the protein. In the ELISA, the optical density of a patient sample exposed to DCLHb-coated wells was compared with that of the patient sample exposed to carbonate buffer-coated wells; an optical density ratio of 1.4 was established for discriminating between a positive (reactive) or negative DCLHb antibody response. To date, all of the more than 300 patient specimens (preinfusion and postinfusion) from clinical trials have exhibited a ratio of less than 1.4, confirming the lack of preexisting antibodies to DCLHb and clearly showing the absence of DCLHb antibodies after exposure to this new biologic entity. There has been no requirement for use of the confirmatory Western blot assay. Taken together, the results from this study indicate DCLHb is not immunogenic in humans at doses evaluated clinically.

Absence of Immunogenicity of Diaspirin Cross-Linked Hemoglobin in Humans

AbstractDiaspirin cross-linked hemoglobin (DCLHb) is an intramolecularly cross-linked hemoglobin-based oxygen carrier being developed as a therapy for acute blood loss. We report here the absence of immunogenicity of DCLHb in patients enrolled in phase II and III clinical trials of DCLHb. Two very sensitive immunoassays, an enzyme-linked immunosorbent assay (ELISA) and a Western blot assay, were developed and validated for this assessment. The DCLHb-antibodies used in these assays were raised in monkeys, had similar affinities for DCLHb and native human hemoglobin (SFHb), and showed cross-reactivity for subunits of DCLHb and SFHb on the Western blot, suggesting that these antibodies were elicited as a xenogenic response to the protein. In the ELISA, the optical density of a patient sample exposed to DCLHb-coated wells was compared with that of the patient sample exposed to carbonate buffer-coated wells; an optical density ratio of 1.4 was established for discriminating between a positive (reactive) or negative DCLHb antibody response. To date, all of the more than 300 patient specimens (preinfusion and postinfusion) from clinical trials have exhibited a ratio of less than 1.4, confirming the lack of preexisting antibodies to DCLHb and clearly showing the absence of DCLHb antibodies after exposure to this new biologic entity. There has been no requirement for use of the confirmatory Western blot assay. Taken together, the results from this study indicate DCLHb is not immunogenic in humans at doses evaluated clinically.

Dose-dependent effect of diaspirin cross-linked hemoglobin on regional blood circulation of severely hemorrhaged rats.

Diaspirin cross-linked hemoglobin (DCLHb), a hemoglobin-based blood substitute, has been found to improve systemic hemodynamics, cutaneous oxygen tension, and normalization of blood lactate levels and acid-base equilibrium after hemorrhage in animals. The present study was conducted to determine the dose-dependent effect of a 10% solution of DCLHb (20, 50, and 100% of shed blood volume; SBV) on regional blood circulation in hemorrhaged rats. Hemorrhage was induced in urethane-anesthetized rats by bleeding them at a rate of approximately .5 to 1 mL/min until a mean arterial pressure of 35-40 mmHg was achieved. This was maintained for up to 90 min to reach a base deficit of more than -12 mmol/L. Hemorrhage significantly decreased oxygen consumption, mean arterial pressure, cardiac output, stroke volume, and regional blood circulation, but increased total peripheral resistance. The vehicle Ringer's lactate (RL at 20% of SBV, intravenously) did not produce any improvements in oxygen consumption, base deficit, systemic hemodynamics, and regional blood circulation. DCLHb increased oxygen consumption, decreased base deficit, and produced significant improvements in systemic hemodynamics and regional blood flow in a dose-dependent manner. The increase in blood flow was highly significant until 60 min, but was less marked at 120 min, after resuscitation with DCLHb. Resuscitation with RL (300% of SBV) significantly improved systemic and regional blood circulation. However, the improvement was greater after resuscitation with DCLHb (50 or 100% of SBV) as compared with RL at 300% SBV. DCLHb in the dose of 50% of SBV produced maximal resuscitative effects, which were comparable to a DCLHb dose of 100% of SBV. The effect of DCLHb at 50% of SBV on renal cortical blood perfusion, concentration of moving red blood cells (CMBC), and blood velocity was also studied using laser Doppler flowmetry. Hemorrhage produced a decrease in renal cortical blood perfusion (85.3%), which was due to a decrease in the CMBC (61.0%) and their velocity (64.2%). Resuscitation with the RL did not produce any improvement in renal cortical perfusion. However, resuscitation with DCLHb significantly increased renal cortical perfusion (364.7%) due to an increase in both CMBC (123.4%) and their velocity (109.9%). It is concluded that DCLHb in a dose of 50% of SBV produces maximal improvement in regional blood circulation of hemorrhaged rats.

Nimodipine inhibits the pressor activity of diaspirin-crosslinked hemoglobin (DCLHb) in the rat.

Impaired nitric oxide (NO) activity is associated with an increase in blood pressure in rats. Voltage-regulated calcium channels are believed to participate in this hemodynamic event. To further test this hypothesis, we examined the effect of nimodipine and verapamil (calcium antagonists) on the pressor activity of diaspirin-crosslinked hemoglobin (DCLHb), a well-known NO scavenger, in anesthetized rats. Nimodipine, the most potent of the two calcium antagonists used, was also tested against phenylephrine (alpha1-adrenoceptor agonist). The pressor effect of DCLHb was reduced markedly by nimodipine and verapamil, whereas that elicited by phenylephrine, particularly the tonic phase of its pressor response, was resistant to blockade by nimodipine. The bradycardia and tachycardia associated with the pressor effects of DCLHb and phenylephrine, respectively, were not affected by nimodipine. The pressor effect elicited by DCLHb and its alteration by nimodipine were also examined in rats pretreated with 100% O2. This treatment was found to potentiate the pressor effect of DCLHb. However, this synergism did not impair the inhibitory action of nimodipine towards the pressor activity of DCLHb. Altogether these results suggest that the pressor activity of DCLHb in our animal model might involve the participation of voltage-regulated calcium channels.

Influence of sepsis on the plasma elimination pharmacokinetics of diaspirin crosslinked hemoglobin in rats.

Septic shock is characterized by abnormalities in microcirculatory O2 delivery (QO2) and profound tissue O2 debt. Administration of crosslinked hemoglobin may be a means of augmenting the QO2 and tissue O2 availability. Sepsis is associated with hemodynamic and metabolic alterations which may affect the pharmacokinetics of crosslinked hemoglobin. The objective of this study was to determine the effect of sepsis on the plasma elimination of diaspirin crosslinked hemoglobin (DCLHb). Twenty-four hours after the induction of sepsis by cecal ligation and perforation, septic (n = 9) and sham rats (n = 8) received an intravenous infusion of 300 mg of DCLHb and arterial blood samples were taken at regular intervals to determine free plasma hemoglobin concentration. DCLHb elimination in septic and sham rats was consistent with first-order elimination kinetics. The half life (t1/2) for septic rats was 4.2 +/- 0.7 h and was significantly shorter than the t1/2 of non-septic rats (5.4 +/- 0.9 h). In all rats, free plasma hemoglobin returned to basal levels by 24 hours after DCLHb administration. The volume of distribution for DCLHb in the septic and non-septic rats was not significantly different and suggests that DCLHb is not influenced by altered gut permeability. Despite significant changes in some elimination parameters the differences were small. Consequently, dosing regimens for this compound may not need to be altered in sepsis.

The effects of diaspirin crosslinked hemoglobin (DCLHb) on oxygenation, perfusion and resuscitation: preclinical experience

The effects of diaspirin crosslinked hemoglobin (DCLHb) on oxygenation, perfusion and resuscitation: preclinical experience

Experimental subarachnoid hemorrhage in rats: effect of intravenous alpha-alpha diaspirin crosslinked hemoglobin on hypoperfusion and neuronal death.

Hemodilution with diaspirin crosslinked hemoglobin (DCLHb) ameliorates occlusive cerebral ischemia. However, subarachnoid hemoglobin has been implicated as a cause of cerebral hypoperfusion. The effect of intravenous DCLHb on cerebral perfusion and neuronal death after experimental subarachnoid hemorrhage was evaluated. Rats (n = 48) were anesthetized with isoflurane and subarachnoid hemorrhage was induced by injecting 0.3 ml of autologous blood into the cistema magna. Each animal received one of the following regimens: Control, no hematocrit manipulation; DCLHb, hematocrit concentration decreased to 30% with DCLHb; or Alb, hematocrit concentration decreased to 30% with human serum albumin. The experiments had two parts, A and B. In part A, after 20 min, cerebral blood flow (CBF) was assessed with 14C-iodoantipyrine autoradiography. In part B, after 96 h, in separate animals, the number of dead neurons was determined in predetermined coronal sections by hematoxylin and eosin staining. Cerebral blood flow was greater for the DCLHb group than for the control group; and CBF was greater for the Alb group than the other two groups (P < 0.05). In one section, CBF was 45.5 +/- 10.9 ml x 100 g(-1) x min(-1) (mean +/- SD) for the control group, 95.3 +/- 16.6 ml x 100 g(-1) x min(-1) for the DCLHb group, and 138.1 +/- 18.7 ml x 100 g(-1) x min(-1) for the Alb group. The number of dead neurons was less in the Alb group (611 +/- 84) than in the control group (1,097 +/- 211), and was less in the DCLHb group (305 +/- 38) than in the other two groups (P < 0.05). These data support a hypothesis that hemodilution decreases hypoperfusion and neuronal death after subarachnoid hemorrhage. The data do not support the notion that intravascular molecular hemoglobin has an adverse effect on brain injury after subarachnoid hemorrhage.

Structural characterization of human hemoglobin crosslinked by bis(3,5-dibromosalicyl) fumarate using mass spectrometric techniques.

Diaspirin crosslinked hemoglobin (DCLHb) was analyzed by mass spectrometric-based techniques to identify the protein modifications effected by the crosslinking reaction with bis(3,5-dibromosalicyl) fumarate. DCLHb consists of two principal components. These components were isolated by size-exclusion chromatography and identified by measurement of their molecular weight using electrospray mass spectrometry and subsequent peptide mass mapping and mass spectrometric sequence analysis of their individual digests. Three major RP-HPLC fractions were observed from the major hemoglobin in DCLHb. Their MWs matched the MW of heme, intact hemoglobin beta-chain, and two hemoglobin alpha-chains crosslinked by a fumarate moiety, respectively. The minor HPLC peaks of DCLHb were also separated, and characterized by mass spectrometric methods. These minor components revealed additional details of the structural nature of covalent modification of DCLHb.

The effects of diaspirin-crosslinked hemoglobin on the assessment of immunohematology profiles.

Extensive studies have been conducted on the in vitro effects of diaspirin-crosslinked hemoglobin (DCLHb) in biochemical, hematologic, hemostatic, and blood banking (immunohematologic) methods. The absence of red cell antigens or plasma and/or serum antibodies allows DCLHb to be used as "universal-donor" material. This study evaluates the effects of DCLHb on the accurate assessment of the immunohematologic profile (ABO and Rh blood grouping, antibody screen, and crossmatching). DCLHb, 7.4 g per dL in an electrolyte solution, was mixed in vitro with human whole blood, representing the blood types A Rh-positive. A Rh-negative, B Rh-positive, B Rh-negative, O Rh-positive, O Rh-negative, and AB Rh-positive. Two concentrations of DCLHb were tested: 10-percent (0.74 g/dL) and 30-percent (2.22 g/dL). Controls were prepared by adding a 5-percent albumin solution to aliquots of whole blood in volumes equivalent to those used in preparing the DCLHb dilutions. Serum and/or red cell suspensions from these admixed samples were analyzed for their ABO and Rh blood groups, the presence of unexpected antibodies (antibody screen), and compatibility in crossmatch testing. DCLHb added to whole blood in vitro had no effect on the accurate interpretation of the immunohematologic profile. DCLHb does not appear to inhibit the true response or crossreact in the analysis of blood grouping, antibody screening, or crossmatching. In addition, the red color of DCLHb (up to 2.22 g/dL) did not obscure the visual reading for agglutination.

Effects of diaspirin-cross-linked hemoglobin (dclhb tm) on local tissue oxygen tension in striated skin muscle: An efficacy study in the hamster

Using the dorsal skin fold chamber model in the hamster, we analyzed local tissue partial oxygen pressure (PO 2) in the striated skin muscle under nonischemic and postischemic conditions with a Clark-type multiwire oxygen surface electrode. Hypervolemic infusion (500 mg · kg −1 IV) or isovolemic exchange transfusion (3.3 gm · kg −1 IV; hematocrit 30%) with diaspirin-cross-linked hemoglobin (DCLHb) resulted in a slight decrease of the mean value of the local tissue PO 2 (mm Hg) 1 hour after administration. Concomitantly, the frequency distribution curves of local tissue PO 2 values were found to be more narrow (fewer values > 25 mm Hg and < 10 mm Hg). Resuscitation from severe hemorrhagic shock (bleeding of 33 ml · kg −1 at 0.4 ml · min −1) with autologous blood (AuB), Dx-60, or DCLHb TM led to an increase of mean tissue PO 2 values by 4.2-fold ( p < 0.05 versus Dx-60), 1.9-fold, and 3.7-fold ( p < 0.05 versus Dx-60), respectively, 2 hours after resuscitation. The reduction of tissue hypoxia (0-5 mm Hg) was significant only in the AuB- and DCLHb TM-treated animals. This study indicates that DCLHb TM effectively reverses tissue hypoxia after resuscitation from severe hemorrhagic shock by inducing a more homogeneous distribution of the local tissue PO 2 levels.

Role of ET and NO in resuscitative effect of diaspirin cross-linked hemoglobin after hemorrhage in rat.

Diaspirin cross-linked hemoglobin (DCLHb) is a hemoglobin-based therapeutic agent that produces significant cardiovascular effects, possibly due to its actions on vasoactive substances, such as endothelin (ET) and nitric oxide (NO). We have studied the modulation of cardiovascular effects of DCLHb by an NO synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), and an ETA-receptor antagonist, FR-139317, in hemorrhaged rats. Control rats resuscitated with vehicle [Ringer lactate (RL), 4 ml/kg iv] did not show any improvement in O2 consumption, base deficit, systemic hemodynamics, or regional blood flow after hemorrhage, and the rats survived for < 70 min. Administration of DCLHb (400 mg/kg iv) significantly improved O2 consumption, base deficit, systemic hemodynamics, and regional blood circulation after resuscitation, and the rats survived for > 120 min after hemorrhage. Plasma ET-1 and guanosine 3',5'-cyclic monophosphate (cGMP) concentrations increased after hemorrhage. DCLHb produced an increase in ET-1 and decreased cGMP concentrations in plasma. Pretreatment with L-NAME (10 mg/kg iv) or FR-139317 (4 mg/kg iv) attenuated the DCLHb-induced improvement in survival time, base deficit, systemic hemodynamics, and regional blood circulation. L-NAME (10 mg/kg iv) per se did not produce any resuscitative effect; therefore the NO mechanism may not be contributing toward the efficacy of DCLHb in hemorrhaged rats. However, FR-139317 attenuated the efficacy of DCLHb; therefore an increase in plasma ET-1 concentration by DCLHb may be contributing toward the efficacy of DCLHb in hemorrhage. Hemorrhage-induced increase in cGMP levels could be attenuated by L-NAME, but L-NAME was not effective in resuscitation of hemorrhaged rats, indicating a lack of role of NO in resuscitation. It is concluded that the ET mechanism is more important in the beneficial effect of DCLHb than the NO mechanism in hemorrhage.

Effects of polymerization on the hypertensive action of diaspirin cross-linked hemoglobin in rats

It is believed that the hypertensive effect of diaspirin crosslinked hemoglobin, a viable blood substitute, can be resolved by polymerization, which reduces the diffusion of this derivative into the interstitial space between nitric oxide-producing endothelium and the target vascular smooth muscle. We studied the systemic and renal responses to infusion of three cell-free human hemoglobins in anesthetized isovolemic rats: unmodified (HbAO), crosslinked (α-DBBF), and polymerized crosslinked (poly α-DBBF). HbAO produced a significant increase in mean arterial blood pressure (MAP) throughout the 60-minute infusion. α-DBBF, on the other hand, produced a more marked and prolonged increase in MAP over 120 minutes. Only a moderate increase in MAP was observed in rats after a 30-minute infusion with poly α-DBBF. The extent of renal insufficiency produced by these proteins, as determined by the glomerular filtration rate, was in the following order: HbAO > poly α-DBBF > α-DBBF. Infusion of poly α-DBBF, under hypovolemic but not isovolemic conditions in rats, produced an increase in heart rate, cardiac output, and stroke volume and a decrease in total peripheral resistance after 60 minutes. Chemical polymerization to increase the size of α-DBBF does not appear to improve its hemodynamic properties in rats, especially under partial exchange transfusion, a more clinically relevant indication for a hemoglobin-based blood substitute.

Resuscitation of pulmonary contusion

To determine the impact of a vasoactive red cell substitute, diaspirin cross-linked hemoglobin, on respiratory derangements after traumatic lung injury. Randomized, controlled animal experiment. Large-animal laboratory. Mechanically ventilated, anesthetized young Yorkshire male swine (15 to 20 kg). Pigs (n = 6/group) received two pneumatic blasts to the right thoracic cage at baseline, were hemorrhaged 30 mL/kg from t = 0 to 20 mins, resuscitated with 0.9% saline (group 1, 90 mL/ kg) or diaspirin cross-linked hemoglobin (group 2, 15 mL/kg) from t = 20 to 40 mins, and then observed to t = 240 mins. Serial pulmonary and systemic hemodynamic measurements, total thoracic compliance assessment, spiral three-dimensional computed tomography scan, and lung weights (n = 3/group) were used to assess lesion size and lung water. Mean arterial pressure was restored in both animal groups. Mean pulmonary arterial pressure was significantly higher after resuscitation in animals receiving the red cell substitute. Oxygenation worsened mildly in both groups. Compliance diminished in both groups but was significantly worse at the end of the experiment in animals infused with diaspirin cross-linked hemoglobin. Right lung weights and right thoracic computed tomography scan volume were higher with diaspirin cross-linked hemoglobin than with saline. After pulmonary contusion, resuscitation with diaspirin cross-linked hemoglobin led to pulmonary hypertension, greater pulmonary contusion lesion size, and stiffer lungs in this porcine model.

Actions of diaspirin cross-linked hemoglobin on isolated rat and dog vessels

The objectives of these studies were to investigate the responses of isolated blood vessels from rats and dogs to the administration of diaspirin cross-linked hemoglobin (DCLHb) and to determine the mechanisms of these responses. Isolated vascular rings (3 to 5 mm) were suspended at optimal passive tension in Krebs-filled (37° C) tissue baths and bubbled with 95% O 2-5% CO 2, and isometric tension was recorded. With the vessels under basal conditions increasing concentrations of DCLHb (10 -8-3 × 10 -6 mol/L) were added. DCLHb addition was repeated during a submaximal contraction with norepinephrine and again during acetylcholine relaxation. The effects of the nitric oxide synthase inhibitor L-nitro arginine (10 -5 mol/L) on the responses to DCLHb were also determined. Dog vessels developed very little tension (1% to 5% of norepinephrine maximum), whereas rat arteries contracted between 9% and 15% when exposed to DCLHb under basal conditions. However, both the dog and rat vessels developed significant tension to DCLHb when they were precontracted (5% to 54%) and also when they were relaxed with acetylcholine (21% to 93%). L-nitro arginine eliminated the contractile responses to DCLHb but did not cause contraction of any of the vessels under basal conditions. We conclude that in this model the mechanism of DCLHb-induced contractions of in vitro dog and rat vessels is dependent on interference with nitric oxide. This is similar to the mechanism of DCLHb action in isolated pig vessels reported previously. Differences in responses of dog, rat, and pig vessels under basal conditions in vitro are the result of active generation of nitric oxide by pig but not by dog or rat vessels.

Alpha-alpha diaspirin crosslinked hemoglobin, nitric oxide, and cerebral ischemic injury in rats.

Prior studies indicate that alpha-alpha diaspirin crosslinked hemoglobin (DCLHb) decreases cerebral ischemia. One mechanism whereby DCLHb may ameliorate cerebral ischemia is by binding nitric oxide (NO), which has been implicated as neurotoxic. We assessed the effect of L-NAME (NO synthase inhibitor) and L-arginine (NO substrate) on ischemic brain injury after DCLHb infusion. Rats were randomized to one of the following groups: Control-no hematocrit manipulation; DCLHb-hematocrit decreased to 16% with 10% DCLHb; DCLHb/L-NAME-hematocrit decreased to 16% with DCLHb, and L-NAME given; DCLHb/L-arg-hematocrit decreased to 16% with DCLHb, and L-arginine given. After 90-min of middle cerebral artery occlusion and 4-hr of reperfusion, infarct volume was determined with TTC stain. Infarct volume (mm3, mean +/- SD) was greater in the Control group (142 +/- 16) than the DCLHb (43 +/- 12), DCLHb/L-NAME (45 +/- 14), and DCLHb/L-arg (71 +/- 18) groups (p < 0.05); was greater in the DCLHb/L-arg group than the DCLHb and DCLHb/L-NAME groups (p < 0.05); but was not different between the DCLHb and DCLHb/L-NAME groups. These data indicate that DCLHb decreases ischemic brain injury, and that binding NO may be one mechanism by which DCLHb decreases ischemic brain injury.

Dose Response Effect of Diaspirin Crosslinked Hemoglobin (DCLHb) on Systemic Hemodynamics and Regional Blood Circulation in Rats

Diaspirin crosslinked hemoglobin (DCLHb, Baxter Healthcare Corporation) a hemoglobin-based blood substitute has been found to increase mean arterial pressure (MAP) in a dose limiting manner. The present study was undertaken to determine dose-dependent effects of DCLHb on systemic hemodynamics and regional blood circulation. DCLHb (10% solution) in doses of 133, 400 and 1200 mg/kg i.v. was given to urethane anaesthetized rats. Normal saline (12 ml/kg) served as a control. Cardiovascular parameters were determined using a radioactive microsphere technique. DCLHb in the doses of 133, 400 and 1200 mg/kg i.v. produced a 46%, 67% and 65% increase in MAP, respectively. Total peripheral resistance (TPR) increased significantly with 133 and 400 mg/kg dose, while cardiac output increased significantly with 400 and 1200 mg/kg dose. There was no change in heart rate. A dose of 133 mg/kg of DCLHb produced a significant decrease in blood flow to the musculoskeletal system, kidney and liver. DCLHb in the dose of 400 and 1200 mg/kg significantly increased blood flow to the heart, gastrointestinal tract (GIT), mesentery & pancreas and skin. All doses of DCLHb produced a significant increase in vascular resistance to the musculoskeletal system and liver. DCLHb in the dose of 133 mg/kg increased resistance to the GIT. heart, skin and kidneys, while the dose of 400 mg/kg increased resistance to the kidneys. A dose of 1200 mg/kg decreased coronary vascular resistance. It is concluded that cardiovascular effects appear to be different with higher (1200 mg/kg) and lower (133 mg/kg) doses of DCLHb.