Effect of haemodilution with diaspirin cross-linked haemoglobin on the oxygen reserve in a rodent model of surgical blood loss.

The efficacy of pre-operative haemodilution is limited by the reduction in haemoglobin concentration. Acellular haemoglobin-based oxygen carriers provide an alternative to colloid as a haemodiluent, potentially extending the safe limits of this procedure. The aim of this investigation was to determine whether haemodilution with a cross-linked haemoglobin solution, diaspirin cross-linked haemoglobin solution (DCLHb), would enhance the oxygen reserve compared to pentastarch. Sprague Dawley rats were placed in a metabolic box to directly measure systemic oxygen consumption (VO2). Rats were randomized to be haemodiluted to a cellular haemoglobin of 80 g L(-1) with either DCLHb or pentastarch. Oxygen reserve was assessed during isovolemic haemorrhage by determining the critical oxygen delivery (DO2crit) and haemoglobin concentration at the point of oxygen supply dependency (OSD). Following haemodilution and for the duration of the experiment, cardiac index (CI) was significantly lower and systemic vascular resistance was significantly higher in the DCLHb than the pentastarch group. The DO2crit (3.2 +/- 0.4 mL minAg(-1) and 3.4 +/- 0.5 mL minAg(-1), DCLHb versus pentastarch) and cellular haemoglobin concentration (51 +/- 9 g L(-1) and 48 +/- 9 g L(-1)), at which rats entered OSD were similar in both groups. Total haemoglobin concentration (cellular and plasma DCLHb) and arterial oxygen content were significantly higher in the DCLHb group (total haemoglobin, 66 +/- 8 g L(-1) and arterial content, 9.2 +/- 1.4 mL dL(-1)) compared to the pentastarch group (total haemoglobin, 48 +/- 9 g L(-1) and arterial content, 7.3 +/- 1.4 mL dL(-1)). Oxygen extraction ratios increased from baseline levels to 0.53 +/- 0.07 and 0.56 +/- 0.1, for the DCLHb and pentastarch groups, respectively, and were not significantly different. The increase in arterial oxygen content from DCLHb in plasma was offset by the decrease in CI observed in this group. Plasma DCLHb did not extend the limits of haemodilution beyond the capacity of the cellular haemoglobin concentration.

Effect of prophylactic transfusion of stored RBCs on oxygen reserve in response to acute isovolemic hemorrhage in a rodent model.

BACKGROUND: The storage of RBCs results in a time-related decline in 2,3 DPG that may reduce the ability to unload oxygen (O(2)) to tissue. The objective of this study was to compare the effect that transfusion of stored 2,3 DPG-depleted rat blood (7 days in CPDA-1) had on the O(2) reserve in conscious rats, with that of the transfusion of fresh blood (<2-hour storage). STUDY DESIGN AND METHODS: Anemic rats (Hb, 80 g/L) received either fresh packed RBCs or stored RBCs to raise Hb levels to 140 g per L. They then underwent isovolemic hemorrhage mimicking surgical blood loss to the point of O(2) supply dependency (OSD). Critical O(2) delivery (DO(2)crit), Hb concentration, and O(2) extraction at OSD were measured in a metabolic chamber. RESULTS: After transfusion, RBC DPG decreased by 50 percent in the stored-blood group, and the p50 value decreased by 5 mmHg (32.1 +/- 2.5 mmHg vs. 37.5 +/- 3.0). DO(2)crit was similar in the two groups (fresh blood: 2.79 +/- 0.44 mL/min x g(-1); stored blood, 2.99 +/- 0.76 mL/min x g(-1)). The critical Hb concentration at DO(2)crit was higher in the stored-blood group (44 +/- 4 g/L) than in the fresh-blood group (38 +/- 5 g/L); the cardiac index and O(2) extraction ratio in the two groups were not different. Under conditions of severe normovolemic anemia in rats, depletion of DPG and a decrease in p50 had only minor effects on the O(2) reserve. At OSD, under these conditions, O(2) consumption is not limited by diffusion. CONCLUSION: The physiologic impact of DPG depletion in transfused stored blood on oxygen availability in normal rats appears to be small and may be clinically inconsequential.

A comparison of biochemical and functional alterations of rat and human erythrocytes stored in CPDA-1 for 29 days: implications for animal models of transfusion.

Animal models of transfusion are employed in many research areas yet little is known about the storage-related changes occurring in the blood used in these studies. This study assessed storage-related changes in red blood cell (RBC) biochemistry, function and membrane deformability in rat and human packed RBCs when stored in CPDA-1 at 4 degrees C over a 4-week period. Human blood from five volunteers and five bags of rat RBC concentrates (five donor rats per bag) were collected and stored at 4 degrees C. RBC function was assessed by post-transfusion viability and the ability to regenerate adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (DPG) when treated with a rejuvenation solution. Membrane deformability was determined by a micropipette aspiration technique. ATP in rat RBCs declined more rapidly than human RBCs; after 1 week rat ATP fell to the same level as human cells after 4 weeks of storage (rat, 2.2 +/- 0.2 micromol g(-1) Hb; human, 2.5 +/- 0.3 micromol g(-1) Hb). Baseline DPG concentrations were similar in rat and human RBCs (16.2 +/- 2.3 micromol g(-1) Hb and 13.7 +/- 2.4 micromol g(-1) Hb) and declined very rapidly in both species. Human RBCs fully regenerated ATP and DPG when treated with a rejuvenation solution after 4 weeks of storage. Rat RBCs regenerated ATP but not DPG. Post-transfusion viability in rat cells was 79%, 26% and 5% after 1, 2 and 4 weeks of storage, respectively. In rats, decreased membrane deformability became significant (- 54%) after 7 days. Human RBC deformability decreased significantly by 34% after 4 weeks of storage. The rejuvenation solution restored RBC deformability to control levels in both species. Our results indicate that rat RBCs stored for 1 week in CPDA-1 develop a storage lesion similar to that of human RBCs stored for 4 weeks and underscores significant species-specific differences in the structure and metabolism of these cells.

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 red cell storage lesion and its implication for transfusion.

Storage of red blood cells in preservative medium is associated with metabolic, biochemical and molecular changes to erythrocytes collectively referred to as the "storage lesion." In addition to corpuscular injury, bioreactive substances including cytokines and lipids accumulate in the medium during storage. We review evidence for those storage related changes and potential clinical implications for red blood cell transfusion.

Validation of transit-time ultrasound flow probes to directly measure portal blood flow in conscious rats.

Direct measurement of portal venous blood flow is technically difficult, yet crucial for accurate assessment of liver hemodynamic and metabolic functions. The aim of this investigation was to assess the feasibility of implanting transit-time ultra-sound (TTUS) perivascular flow probes on the portal vein of the rat and to validate this technique as a means of directly measuring portal blood flow in conscious rats. A TTUS flow probe was implanted on the portal veins of 10 rats. One week later, portal flow was measured under basal conditions in these rats by TTUS probes and after pharmacological manipulation of portal flow by intravenous injections of Glypressin or infusions of adenosine while the rats were conscious. Portal flow was simultaneously measured in the same rats using radioactive microspheres. Basal systemic hemodynamics, regional blood flows to splanchnic organs, and portal blood pressure were not significantly modified by the presence of the probe on the portal vein compared with a control group of rats not instrumented with flow probes. Basal portal flows measured by the TTUS and microsphere techniques were not different (20.6 +/- 2.6 and 17.6 +/- 1.3 ml/min). After Glypressin, portal flows measured by the TTUS and microsphere techniques were 12.3 +/- 2.9 and 9.3 +/- 1.9 ml/min and, in response to adenosine, increased to 27.2 +/- 3.4 and 31.3 +/- 4.1 ml/min. There was no significant difference between the TTUS and microsphere flows. Both the relationship between absolute flows and the relationship between changes in flows measured by the two techniques were linear with slopes approaching 1.0. Thus TTUS flow probes can be used to directly measure portal flow from the portal vein in conscious rats. This methodology is as effective as the standard technique of radioactive microspheres. More importantly, the TTUS technique allows for continuous direct measurement of portal flow and eliminates the hazards and sources of error associated with the radioactive microsphere technique.

Validation of 1- and 2-mm transit-time ultrasound flow probes on mesenteric artery and aorta of rats.

The aim of this investigation was to validate and calibrate the 1- and 2-mm transit-time ultrasound, perivascular flow probes on the superior mesenteric artery and aorta of the rat by using an in vivo and in situ calibration system. The baseline blood flows measured by the flowmeter zero-flow function and the true-zero flow measurement, after complete occlusion of the vessels, were not different. The probes were then calibrated, in situ, by timed-volume measurements with a blood/saline pump-perfused circuit. The relationship between measured flow and true flow was linear for both probes. The slope of the 1-mm probe data was not significantly different from the line of identity, whereas the 2-mm probe data slightly overestimated the true flow (slope = 1.2, y-intercept = -3.2 ml/min). The slope of the middle region of the 2-mm probe data, however, was not significantly different from the line of identity, suggesting that the 2-mm probe has a window of accuracy over this range of flows. Progressive elimination of the low and high data points varying the most from the line of identity and reanalysis of the data revealed that the window of accuracy for the 2-mm probe, under our experimental conditions, was between 10.4 and 45.1 ml/min.

Hepatic blood volume responses and compliance in cats with long-term bile duct ligation.

Hepatic capacitance responses were compared in sham-operated and 14-day bile duct-ligated cats under pentobarbital anesthesia. Both groups were subjected to splenectomy and had the anterior hepatic nerve plexus sectioned to allow stimulation; the posterior plexus was intact. Blood volume compensation for hemorrhage was reduced in the bile duct ligation group compared with the control group: The liver compensated for 20.1% and 10.6% of blood loss, respectively. Portal hypertension did not exist and hepatic compliance was unaltered despite the presence of severe biliary hyperplasia, portal tract distortion and fibrosis. The capacitance responses to hemorrhage in the bile duct ligation group were accounted for entirely by a passive compliant response to reduced portal pressure, whereas the sham surgery group showed an additional active component. Responses of liver volume and venous resistance to norepinephrine were normal, but responses to nerve stimulation were reduced. The nerve dysfunction was not universal; reflex arterial blood pressure response to carotid occlusion was normal. The data suggest that hepatic blood volume responses to hemorrhage is mediated by passive compliant responses to reduced portal pressure (stressed volume) and active responses (unstressed volume) to nerve stimulation but that long-term bile duct ligation produces selective hepatic neuropathy resulting in loss of the active component.

Expression of vascular escape: conductance or resistance?

Vascular escape is that phenomenon whereby a tachyphylaxis occurs in the vasoconstriction of an arteriole to a constant sympathetic stimulation. Vascular escape, in vivo, is primarily a blood flow event. Calculated resistance, as an index of vascular tone, does not consistently describe the responses of the arterioles undergoing vascular escape. Conductance, which is the inverse of resistance, obviates several of the errors produced by the use of resistance. In this study, we illustrate this issue using hypothetical and experimental data. Escape responses were calculated in terms of resistance and conductance and plotted against blood flow escape responses. Resistance escape responses were nonlinearly related to blood flow escapes and overestimated vascular escape with both hypothetical and experimental data. Conductance escape responses were linearly related to flow escape responses and consistently described vascular escape. We therefore conclude that conductance is a better index of vascular tone to express vascular escape.

Glucagon pharmacodynamics and modulation of sympathetic nerve and norepinephrine-induced constrictor responses in the superior mesenteric artery of the cat.

The aim of this study was to assess whether the pancreatic peptide glucagon was capable of inhibiting nerve- and norepinephrine-induced vasoconstrictor responses in the superior mesenteric artery of the anesthetized cat. Intra-arterial dose-response curves for glucagon and norepinephrine were analyzed by nonlinear regression to estimate the maximal response (maximal dilation, 163%; maximal constriction, 110%) in terms of percent change in superior mesenteric artery conductance and dose of glucagon or norepinephrine required to produce 50% of the maximal response (0.98 and 0.38 micrograms/kg/min, respectively). Constrictor responses (3-min duration) were only weakly inhibited by glucagon. Peak constrictor responses induced by low-dose i.a. infusions of norepinephrine were significantly inhibited (39%) by the high dose of glucagon, whereas the high-dose norepinephrine peak constrictor responses were unaffected by any dose of glucagon. Intermediate and high doses of glucagon significantly inhibited the low-frequency (2 Hz) nerve-induced peak constrictions (19% and 34%, respectively). The higher frequency (6 Hz) nerve-induced peak constrictor responses were not significantly affected by glucagon. Vascular escape from nerve- and norepinephrine-induced peak constrictor responses was not related to the degree of initial constriction nor were they affected by glucagon. Glucagon levels produced by our i.a. infusions were estimated to be well outside the pathophysiological range. We conclude that glucagon is not an effective inhibitor of constrictor responses in the superior mesenteric artery and is unlikely to have such an effect at physiological levels.

The effect of glucagon on vasoconstriction and vascular escape from nerve- and norepinephrine-induced constriction of the hepatic artery of the cat.

Glucagon, in the anesthetized cat, was capable of dilating the hepatic artery to the same extent and in a dose-dependent manner when administered directly into the hepatic artery or into the portal vein. Portal venous infusions of glucagon did not inhibit nerve- or norepinephrine-induced vasoconstriction of the hepatic artery in contrast to previous reports in the dog. Rather, at certain doses, glucagon mildly potentiated the vasoconstriction induced by both constrictor stimuli. Vascular escape from nerve- and norepinephrine-induced constrictor responses was found to be inhibited by glucagon in a dose-dependent manner. Glucagon infusion is the first intervention reported to modulate vascular escape in the hepatic artery. Owing to its similar effects on nerve- and exogenous norepinephrine-induced responses, glucagon appears to be acting at a postsynaptic site. Therefore, we suggest that in the cat, glucagon is not an inhibitory modulator of nerve- and norepinephrine-induced vasoconstriction, but rather may potentiate the constrictor response in a postsynaptic manner.

Impact of the hepatic arterial buffer response on splanchnic vascular responses to intravenous adenosine, isoproterenol, and glucagon.

Hepatic arteries (HA) and superior mesenteric arteries (SMA) of cats anesthetized with pentobarbital responded to direct intra-arterial infusion of isoproterenol, adenosine, and glucagon with dose-related vasodilation. In response to intravenous infusion, however, the HA failed to dilate significantly, while the SMA dilated thus elevating portal blood flow. The lack of dilation of the HA was due to the HA buffer response to the elevated portal blood flow, that is, elevation of portal flow causes the HA to constrict. When a clamp was used to return SMA flow to control levels during infusion of the drugs, the HA showed significant dilation to all three agents. Thus, HA vascular responses to i.v. drugs can only be assessed if portal flow is known, since the net effect is dependent upon direct action of the drug on the HA as well as the indirect effect of any drug-induced change in portal flow. None of the agents tested altered the magnitude of the HA buffer response obtained during i.v. infusions, but the effects of other agents on the buffer response remain unknown and must be considered in any tests of i.v. administered drugs. Bolus i.v. injections produce results on the HA flow that are uninterpretable.

Adenosine as putative regulator of hepatic arterial flow (the buffer response).

In anesthetized cats, reduction of portal flow by occlusion of the superior mesenteric artery results in rapid increase in hepatic arterial (HA) flow that compensates for (buffers) 25.5 +/- 2.7% of the decreased portal flow. The hypothesis tested is that adenosine concentration produced near the HA resistance vessels is regulated by washout into portal vessels in intimate contact with the HA. Reduced portal flow leads to accumulation of adenosine and HA dilation. Several criteria for this hypothesis are met. First, adenosine is a potent dilator of the HA. Second, portal blood has access to HA resistance vessels as shown by a marked dilator effect of adenosine infused into the portal vein; it is therefore possible for adenosine produced locally to diffuse into portal blood. Third, dipyridamole potentiated the dilator response to adenosine as well as potentiating the buffer response from a 23% compensation for reduced portal flow to 34%. Fourth, 1-methyl-3-isobutylxanthine (MIX) antagonized exogenous adenosine and reduced the buffer response from 19% down to 5%. These data strongly support the hypothesis that the hepatic arterial buffer response is mediated by local concentrations of adenosine that are controlled by the rate of washout into portal blood.