Isovolemic Replacement Research Articles

Hemoglobin extravasation in the brain of rats exchange-transfused with hemoglobin-based oxygen carriers

Haemoglobin (Hb)-based oxygen carriers are under consideration as oxygen therapeutics. Their effect on apoptosis is critical, because the onset of pro-apoptotic pathways may lead to tissue damage. MP4OX, a polyethylene glycol-conjugated human Hb preserves the baseline level of neuron apoptosis with respect to sham. Here we develop a method for measuring Hb extravasation in brain. We exchange transfused rats by haemorrhaging 50% of their blood with simultaneous, isovolemic replacement with Hextend (negative control), MP4OX, or αα-cross-linked Hb. Animals were sacrificed 2 h after transfusion, brain tissue was harvested and processed for double-staining immunofluorescence, whereby Hb ? chain and NeuN (a neuron protein) were stained and quantitated. Whereas Hextend did not induce Hb extravasation, in both MP4OX and ??Hb brains Hb molecules were detected outside neurons. The level of extravasated Hb chains was > 3-fold higher in Hb compared to MP4OX. Western blot analysis revealed that the expression levels of protein related to redox imbalance (e.g., Nrf2, iNOS and ERK phosphorylation) were higher in ααHb than MP4OX. In conclusions, higher Hb extravasation in ααHb than MP4OX induces redox imbalance, which causes higher anti-oxidant response. Whereas Nrf2 response may be considered protective, iNOS response appears damaging.

Impact of acellular hemoglobin‐based oxygen carriers on brain apoptosis in rats

Extracellular hemoglobin (Hb)-based oxygen carriers (HBOCs) are under extensive consideration as oxygen therapeutics. Their effects on cellular mechanisms related to apoptosis are of particular interest, because the onset of proapoptotic pathways may give rise to tissue damage. The objective was to assess whether the properties of the Hb that replaces blood during an isovolemic hemodilution would modulate apoptotic-response mechanisms in rat brain and whether such signaling favors cytoprotection or damage. We exposed rats to exchange transfusion (ET; 50% blood volume and isovolemic replacement with Hextend [negative colloid control], MP4OX [PEGylated HBOC with high oxygen affinity], and ααHb [αα-cross-linked HBOC with low oxygen affinity; n=4-6/group]). Sham rats acted as control. Animals were euthanized at 2, 6, and 12 hours after ET; brain tissue was harvested and processed for analysis. In MP4OX animals, the number of neurons that overexpressed the hypoxia-inducible factor (HIF)-1α was higher than in ααHb, particularly at the early time points. In addition, MP4OX was associated with greater phosphorylation of protein kinase B (Akt), a well-known cytoprotective factor. Indeed, the degree of apoptosis, measured as terminal deoxynucleotidyl transferase-positive neurons and caspase-3 cleavage, ranked in order of MP4OX < Hextend < ααHb. Even though both HBOCs showed increased levels of HIF-1α compared to shams or Hextend-treated animals, differences in signaling events resulted in very different outcomes for the two HBOCs. ααHb-treated brain tissue showed significant neuronal damage, measured as apoptosis. This was in stark contrast to the protection seen with MP4OX, apparently due to recruitment of Akt and neuronal specific HIF-1α pathways.

Responses of Amniotic Fluid Volume and Its Four Major Flows to Lung Liquid Diversion and Amniotic Infusion in the Ovine Fetus

We designed experiments to allow direct measurement of amniotic fluid volume and continuous measurement of lung liquid production, swallowing, and urine production in fetal sheep. From these values, the rate of intramembranous absorption was calculated. Using this experimental design, the contribution of lung liquid to the control of amniotic fluid volume was examined. Fetuses were assigned to 1 of 4 protocols, each protocol lasting 3 days: control, isovolemic replacement of lung liquid, supplementation of amniotic fluid inflow by 4 L/day, and supplementation of amniotic inflow during isovolemic replacement of lung liquid. We found no effect of lung liquid replacement on any of the known flows into and out of the amniotic fluid. Although intramembranous absorption increased greatly during supplementation, the amniochorionic function curves were not altered by isovolemic lung liquid replacement. We conclude that lung liquid does not appear to contain a significant regulatory substance for amniotic fluid volume control.

Mathematical analysis of isovolemic hemodilution indicates that it can decrease the need for allogeneic blood transfusion.

The implementation of acute isovolemic hemodilution prior to surgical blood loss is a strategy used in an attempt to diminish the need for or obviate allogeneic transfusion and to avert the potential, attendant complications. Studies examining the efficacy of this technique have produced conflicting results. The present mathematical analysis was undertaken to resolve these conflicts by determining the efficacy of hemodilution and examining the influence of the variables affecting the outcome. Efficacy was defined as the volume of additional blood loss permitted and the volume and number of units of allogeneic blood saved from transfusion. A mathematical analysis evaluated the impact of circulating blood volume and initial and target hematocrits on the efficacy of isovolemic hemodilution. It was assumed that 1) hemodilution was completed before surgical blood loss; 2) transfusion of removed blood was begun when the target hematocrit was reached and lost surgical blood was replaced at a rate that maintained the target hematocrit; 3) allogeneic transfusion was begun after all autologous blood drawn was transfused; 4) normovolemia was maintained; and 5) a unit of allogeneic blood contains 175 mL of red cells. The analysis showed that isovolemic hemodilution can result in substantial additional allowable surgical blood loss that can diminish the need for or obviate allogeneic transfusion of red cells. Larger circulating blood volume, higher initial hematocrits, and lower target hematocrits increase the efficacy of hemodilution. Removal and isovolemic replacement of 1 to 2 units of blood provide minimal potential savings, as does hemodilution to a circulating (target) hematocrit of 30 percent. The extension of hemodilution to a hematocrit of (or below) 20 percent allows a disproportionately greater surgical blood loss and diminishes the need for allogeneic transfusion. It allows, for example, an additional 4.5 L of surgical blood loss, which represents a savings of 4 units of allogeneic blood when a patient with an initial blood volume of 5.0 L and a hematocrit of 45 percent undergoes isovolemic hemodilution to a hematocrit of 15 percent. Isovolemic hemodilution can diminish or in some circumstances eliminate the need for allogeneic transfusion.

Effect of reduced hemoglobin concentration on leg oxygen uptake during maximal exercise in humans

Maximum oxygen uptake (VO2max) is affected by hemoglobin concentration ([Hb]). Whether this is simply due to altered convection of O2 into the muscle microcirculation or also to [Hb]-dependent diffusive transport of O2 out of the muscle capillary is unknown in humans. To examine this, seven healthy volunteers performed four maximal cycle exercise bouts at sea level immediately after 8 wk at altitude (3,801 m, barometric pressure 485 Torr), a sojourn designed to increase [Hb]. The first two bouts were at ambient [Hb] of 15.9 +/- 0.7 g/100 ml breathing 21 or 12% O2 in random order. [Hb] was then decreased to a prealtitude level of 13.8 +/- 0.6 g/100 ml by venesection and isovolemic replacement with 5% albumin in 0.9% saline, and the exercise bouts were repeated. At whole body VO2max, PO2, PCO2, pH, and O2 saturation were measured in radial arterial and femoral venous blood. Femoral venous thermodilution blood flow was determined for calculation of leg VO2. Mean muscle capillary PO2 and muscle diffusing capacity (DO2) were computed by Bohr integration between measured arterial and femoral venous PO2. Averaged over both fractional concentrations of inspired O2, leg VO2 at maximum decreased by 17.7 +/- 4.3% as [Hb] was lowered while leg O2 delivery decreased by 17.5 +/- 2.6% and DO2 decreased by 10.7 +/- 2.7% (all P < 0.05). The relative contributions of decreases in leg O2 delivery and DO2 to the decrease in VO2max were computed to be 64 and 36%, respectively. These findings suggest that [Hb] is an important determinant of O2 diffusion rates into working muscle in humans. Possible mechanisms include 1) dependence of DO2 on intracapillary red blood cell spacing, 2) changes in the total rate of dissociation of O2 from [Hb], and 3) increased red blood cell flow heterogeneity as [Hb] is reduced.

Renal response to metabolic alkalosis induced by isovolemic hemofiltration in the dog

We describe a new model of chloride-depletion alkalosis (CDMA), in which the method of induction of alkalosis does not itself cause a direct alteration in sodium and fluid balance. We have used this model, which is based on hemofiltration techniques in the dog, to study the immediate response of the kidney to the induction of CDMA. Normal dogs maintained with a NaCl-free diet for several days underwent hemofiltration of 50 ml/kg over a 35 minute period. The hemofiltrate was replaced ml for ml with a solution containing sodium and potassium in the same concentrations as found in each animal's plasma water. In control animals, the replacement solution contained chloride and bicarbonate in the same ratio as in the plasma; in the experimental (CDMA) animals the replacement solution contained bicarbonate as the only anion. In the control group, the procedure of hemofiltration coupled with isovolemic replacement caused no appreciable changes in plasma composition, urinary excretion rates, GFR, or tubular handling of bicarbonate. In the CDMA group, 106 +/- 8.4 mEq of chloride were removed in exchange for bicarbonate. A marked metabolic alkalosis resulted, plasma bicarbonate concentration increasing from 21.9 +/- 0.6 to 33.3 +/- 0.6 mEq/liter. The hemofiltration procedure itself, by design, did not alter sodium or fluid balance. Nevertheless, cumulative urinary sodium excretion increased over 2.5 hours by 23.0 +/- 6.4 mEq. A natriuresis of this magnitude is equivalent to a loss of ECF volume of approximately 200 ml. GFR did not change significantly. The rate of tubular reabsorption of bicarbonate increased significantly from 1209 +/- 82 to 1559 +/- 148 mu Eq/min in CDMA animals.(ABSTRACT TRUNCATED AT 250 WORDS)

A method for large volume blood sampling and transfusion in rats.

A new protocol that makes it feasible to withdraw large volumes of whole blood from an individual rat within 1 h or less is described. This method involves the use of indwelling catheters for withdrawal of blood from the inferior vena cava with concurrent isovolemic replacement of whole blood into the superior vena cava. Simultaneity of the transfusion and withdrawal, strict equality of volumes, and a smooth exchange of blood are assured by the use of separate channels of the same multiple-channel peristaltic pump for withdrawal and replacement. Validation experiments using both anesthetized and unanesthetized rats indicate that several responses remain essentially undisturbed during large volume blood sampling; these parameters include blood pressure, heart rate, hematocrit, plasma hormones including insulin and glucagon, plasma glucose levels, and feeding behavior. Considerations of technical and physiological limitations of the protocol, including choice of catheters and catheter placement, pump, sampling parameters, and obtaining donor blood, are discussed.

GLOMERULAR PERMEABILITY IN AUTOLOGOUS IMMUNE COMPLEX (AIC) NEPHRITIS

Glomerular permeability to neutral polydisperse polyvinylpyrrolidone 125I (PVP) was studied in Foreman rats with and without autologous immune complex glomerulonephritis (AIC). The Foreman rat generally develops milder disease than other strains. However, all AIC rats studied were nephrotic with proteinuria in excess of SOmg/24 hrs. PVP clearance (MW 8000-85000) was determined by column chromatography and compared to simultaneous inulin clearance (Cin). Particle diameter (Å) was calculated by deriving RS, radius of equivalent sphere, for each PVP fraction. Clearances were performed without volume expansion, using iso-volemic replacement of blood samples.Cin was the same in AIC and controls. Mean permeability to molecules > 4oÅ was only slightly increased in AIC (p > .5). In contrast, permeability to molecules < 4oÅ was decreased, as previously reported with PVP in idiopathic nephrotic syndrome (INS)1 and with dextran in nephrotoxic serum nephritis.2 cpvp < 25Å approached 100% Cin in controls, whereas this occurred in AIC only at < 15Å (p < .05); i.e. passage of low MW PVP particles in AIC was restricted but not as much as in INS, where CPVP never approached that of Cin. Permeability changes associated with the presence of immune complexes in AIC may obscure the restriction of small MW PVP found when there is foot-process disease alone.1. Robson, A.M., et al: JCI 54:1190, 11/74.2. Chang, R.L.S., et al: JCI 57:1272, 5/76.

Oxygen transport and delivery in rabbits treated with cyanate

Summary Oxygen transport, delivery and utilization were compared in normal and cyanate-treated rabbits made progressively more anemic by isovolemic replacement of blood by a buffered albumin solution. Oxygen delivery, consumption, and utilization did not differ significantly between the two groups of animals at packed cell volumes of over 20% to less than 5% despite the marked left shift in the oxyhemoglobin dissociation curve in the animals treated with cyanate. Since there was no evidence of tissue hypoxia in the latter group, it is concluded that tissue hypoxia will also not be seen in patients treated with smaller doses of sodium cyanate.