Robert L. Conhaim

Resuscitation from hemorrhagic shock with diaspirin cross-linked hemoglobin, blood, or hetastarch.

BACKGROUNDnAn oxygen-transporting hemoglobin solution should be more effective than a nonhemoglobin solution for resuscitation from hemorrhagic shock. A way to evaluate this effectiveness is to determine whether a hemoglobin solution can reverse the base deficit accumulated during hemorrhage at a faster rate than a nonhemoglobin solution. Using this criterion, we compared the resuscitative powers of autologous blood, hetastarch (Het), and diaspirin cross-linked hemoglobin (DCLHb).nnnMETHODSnFifteen sedated, spontaneously breathing sheep (37.5 +/- 10.2 kg) were bled until base deficits fell to -5 to -10 mEq/L, and plasma lactate concentrations rose to 6 to 9 mg/L. The animals were resuscitated with autologous blood (n = 5), Het (n = 5), or DCLHb (n = 5) (3.5-4.0 mL/kg every 15 minutes) until base deficits returned to prehemorrhage baseline.nnnRESULTSnExsanguination to target base deficits required removal of an average of 41.4 +/- 5.5 mL blood/kg (estimated total blood volume, 80 mL/kg). Resuscitation required 18 +/- 3, 38 +/- 2 (different from blood), and 35 +/- 1 (different from blood) mL/kg of autologous blood, Het and DCLHb, respectively, over periods of 78 +/- 8, 163 +/- 10 (different from blood), and 129 +/- 9 minutes (different from blood and different from Het (p < or = 0.05)). Based on regression analysis, autologous blood, Het, and DCLHb corrected the base deficit at rates of, respectively, 0.074 (different from Het (p < or = 0.05)), 0.016, and 0.056 (different from Het (P < or = 0.05)) mEq/L/min.nnnCONCLUSIONSnBased on the rate of base deficit correction and the volume of solution required, autologous blood was the most effective resuscitation solution. However, DCLHb was more effective than Het. DCLHb may be an attractive alternative to blood for resuscitation from hemorrhagic shock.

Hypoxia recruits intrapulmonary arteriovenous pathways in intact rats but not isolated rat lungs.

Intrapulmonary arteriovenous anastomoses (IPAVS) directly connect the arterial and venous circulations in the lung, bypassing the capillary network. Here, we used solid, latex microspheres and isolated rat lung and intact, spontaneously breathing rat models to test the hypothesis that IPAVS are recruited by alveolar hypoxia. We found that hypoxia recruits IPAVS in the intact rat, but not the isolated lung. IPAVS are at least 70 μm in the rat and, interestingly, appear to be recruited when the mixed venous Po(2) falls below 22 mmHg. These data provide evidence that large-diameter, direct arteriovenous connections exist in the lung and are recruitable by hypoxia in the intact animal.

Effects of pentafraction and hetastarch plasma expansion on lung and soft tissue transvascular fluid filtration

BACKGROUNDnHetastarch and pentafraction are high molecular weight starch solutions designed to augment plasma oncotic pressure. Although clinical utilization of hetastarch has been limited by reported coagulation abnormalities, pentafraction is a newer derivative that appears to have few adverse hemostatic effects. We examined the ability of pentafraction to modulate lung and soft tissue transvascular fluid filtration under hypoproteinemic conditions compared with hetastarch and Ringers lactate (LR).nnnMETHODSnAwake, protein-depleted sheep (n = 19) were prepared with lung and soft tissue lymph fistulas, and comparable infusions of 5% pentafraction (n = 6), 6% hetastarch (n = 6), or LR (n = 7) were administered. Plasma and lymph samples were collected during 24-hour period to determine changes in protein concentrations, plasma-to-lymph oncotic gradients, and lung (QL) and soft tissue (QS) lymph flows.nnnRESULTSnQL and QS rose nearly twofold after protein depletion alone. LR infusion increased QL and QS to 8.7 +/- 1.7 and 3.1 +/- 0.6 times normoproteinemic baseline, respectively (p < 0.05). In contrast, hetastarch and pentafraction infusion limited the increase in QL to 4.2 +/- 1.1 and 4.0 +/- 0.8 times normoproteinemic baseline, respectively (p < 0.05 versus LR) and did not significantly increase QS. Hetastarch and pentafraction infusions increase plasma oncotic pressure by nearly 6 mm Hg, which significantly widened the plasma-to-lymph oncotic pressure gradients above preinfusion baseline by 4.7 +/- 0.7 and 3.4 +/- 0.4 mm Hg in lung and 4.6 +/- 0.7 and 3.2 +/- 0.4 mm Hg in soft tissue, respectively (p < 0.05).nnnCONCLUSIONSnBoth hetastarch and pentafraction limit transvascular fluid filtration under hypoproteinemic conditions by augmenting plasma oncotic pressure and the plasma-to-lymph oncotic pressure gradient. Because of fewer adverse hemostatic effects pentafraction may be an improvement over current therapies in critical care fluid management.

Pulmonary capillary sieving of hetastarch is not altered by LPS-induced sepsis.

BACKGROUNDnGram-negative lipopolysaccharide (LPS) has been demonstrated to increase pulmonary capillary permeability as judged by the increased flow of protein-rich lymph from the lungs of sheep infused with LPS. This finding suggests that LPS-injured pulmonary capillaries might be less restrictive than uninjured capillaries to the filtration of large hetastarch molecules. Hetastarch has a broad molecular mass spectrum (35-1,500 kilodaltons (kDa)), and one way to test the restrictiveness of pulmonary capillaries is to measure the size of the largest hetastarch molecules that cross the microvascular barrier and enter the lymph. To evaluate the effects of LPS, we compared hetastarch molecular distributions in the lung lymph of normal and LPS-injured sheep.nnnMETHODSnAdult sheep (38.2 +/- 0.8 kg) were surgically prepared for the collection of lung lymph, with study initiation after a 5- to 7-day recovery period. Hetastarch (6%) was infused (10 mL/kg) 24 hours before study to allow for stabilization of the hetastarch molecular distribution. On the day of study, LPS (Escherichia coli lipopolysaccharide, 2 microg/kg; n = 6) was infused, and plasma and lymph samples were collected for 12 hours. An additional group of animals not infused with LPS (n = 6) served as controls. Hetastarch molecular distributions in plasma and lymph were measured by using high performance size exclusion chromatography.nnnRESULTSnIn control sheep, the largest hetastarch molecules in lymph averaged 861 +/- 18 kDa (mean +/- SEM) (plasma, 1,065 +/- 18 kDa). In LPS-treated sheep, the largest hetastarch molecules in lymph averaged 845 +/- 19 kDa (not significant vs. normal) (plasma, 1,025 +/- 14 kDa). Hetastarch concentrations in plasma and lung lymph of normal sheep, respectively, were 0.61 +/- 0.05% and 0.34 +/- 0.07%. In LPS-treated sheep, hetastarch concentrations in plasma and lymph were 0.56 +/- 0.08 (not significant vs. normal) and 0.29 +/- 0.07, respectively (p < or = 0.05). Lymph concentrations were lower after LPS because of increased lymph flows (19.9 +/- 5.4 mL/30 min, compared with 3.6 +/- 0.8 mL/30 min in normal sheep).nnnCONCLUSIONnOur results suggest that LPS does not alter the diameter of the largest pores perforating the walls of pulmonary capillaries. Rather, the number of these pores in the capillary wall appears to be increased. This increase would explain why lymph flows rise after LPS with little change in the lymph protein concentration. Our results are also consistent with a filtration model in which capillaries are assumed to be perforated by small pores (protein reflection coefficient = 1) as well as large pores (protein reflection coefficient = 0).

Hemoglobin therapeutics in hemorrhagic shock

&NA; Clinical approval is presently being sought for the use of acellular hemoglobin solutions in the treatment of hemorrhagic shock. These novel solutions have oxygen‐transporting properties similar to those of whole blood but do not require blood typing and can be stored frozen. The acellular nature of these solutions gives them properties not possessed by whole blood: they have the pharmacologic properties of a pressor, and they can filter out of the circulation. Whether these additional properties are beneficial or detrimental to the treatment of hemorrhagic shock continues to be debated. Clinical trials on Baxter Healthcares diaspirin cross‐linked hemoglobin (HemAssist; Baxter Healthcare, Round Lake, IL) were recently suspended because of a higher than expected mortality rate in the treatment study group and a lower than expected mortality rate in the control group.

Microthrombus formation may trigger lung injury after acute blood loss.

We showed previously that acute blood loss, without resuscitation, caused marked maldistribution of interalveolar perfusion. Because hemorrhage is a known risk factor for the development of lung injury, the goal of our present studies was to determine if there was a correlation between perfusion maldistribution and the subsequent development of lung injury after blood loss. Specifically, we wanted to know if the perfusion maldistribution might be due to microthrombus formation and/or leukocyte sequestration within the pulmonary microcirculation. We bled rats (30% blood loss) and harvested their lungs 45 min or 24 h later. Lungs were prepared for perfusion distribution analysis, Western blot analysis to measure whole-lung fibrinogen concentrations, and for immunohistochemistry to measure fibrin deposition and leukocyte deposition (CD16 fluorescence). Perfusion was significantly maldistributed at 45 min and 24 h (P < 0.05). At 45 min, whole-lung fibrinogen concentrations were less than half that in controls (P < 0.05), whereas numbers of fibrin microthrombi were 2.5-fold greater than control by 45 min (not statistically significant) and were 4.5-fold greater by 24 h (P = 0.01). Leukocyte deposition was two-fold greater than control by 45 min (not statistically significant) and was 4-fold greater by 24 h (P = 0.02). Fibrin-to-leukocyte nearest-neighbor distances remained unchanged (18.1 [SD, 1.1] &mgr;m) even as the numbers of both increased with time after blood loss. Our results suggest that soluble fibrinogen polymerized to insoluble fibrin within minutes after acute blood loss, which caused perfusion maldistribution and attracted leukocytes. The development of lung injury after blood loss may be a consequence of leukocyte chemoattraction to fibrin microthrombi that seem to form within minutes after blood loss.

Pulmonary transvascular fluid filtration response to hypoproteinemia and hespan infusion

Management of major blood loss utilizing protein-free fluids for volume replacement frequently results in plasma protein depletion and plasma volume expansion. These factors can increase pulmonary transvascular fluid filtration which may lead to life-threatening pulmonary edema. We studied the combined effects of plasma protein depletion and plasma volume expansion on lung lymph flow (QL) in awake sheep prepared with chronic lung lymph fistulae. Animals were first chronically protein-depleted by batch plasmapheresis and then infused for 2 hr with either lactated Ringers (Hypo/LR; n = 7) or 6% hydroxyethyl starch (Hespan) (Hypo/HES; n = 6). Control normoproteinemic animals (Norm/LR; n = 13) only received lactated Ringers. Hypoproteinemia alone resulted in an average 2-fold increase in QL over normoproteinemic baseline levels (P less than or equal to 0.05). Infusion of LR into hypoproteinemic animals caused a 7.9-fold increase in QL (P less than or equal to 0.05). By comparison, HES infusion under similar hypoproteinemic conditions limited the increase in QL to 3.2-fold over baseline. We attributed this reduced rise in QL to Hespans high oncotic pressure, which dramatically widened (by 4-5 mm Hg) the pulmonary-to-lymph oncotic pressure gradient. We did not observe this with LR infusion, or in previous studies employing intravenous infusion of plasma protein. Thus, the oncotic pressure of Hespan appears to significantly limit pulmonary fluid filtration during hypoproteinemia compared to LR. We do not believe that these effects are the results of any changes in microvascular porosity.

Effects of University of Wisconsin and Euro-Collins solutions on interstitial pulmonary edema in isolated rat lungs.

Macromolecules are present in lung preservation solutions to limit liquid filtration out of the pulmonary circulation and minimize pulmonary edema. We tested the effectiveness of these molecules by measuring interstitial edema in rat lungs perfused with macromolecular solutions (University of Wisconsin [UW] solution and Euro-Collins solution supplemented with modified pentastarch [pentafraction, PEN]) or with solutions that lacked macromolecules (UW solution with PEN and Euro-Collins solution.) The lungs were inflated with air and perfused with one of the test solutions, then rapidly frozen and prepared for histological analysis. From tissue sections, we measured cross-sectional areas of pulmonary arteries and veins, and also measured cross-sectional areas of the interstitial spaces surrounding arteries and veins. We then calculated the interstitium-to-vessel cross-sectional area ratio. In lungs perfused with macromolecular solutions these ratios were 0.09+/-0.15 and 0.53+/-0.56 (mean +/- SD) for UW solution and Euro-Collins solutions solution with PEN, respectively (P</=0.05). In lungs perfused with solutions that lacked macromolecules, area ratios were 0.48+/-0.88 and 1.95+/-1.82 for UW solution without PEN and Euro-Collins solution, respectively (P</=0.05). Solutions containing PEN caused less interstitial expansion than their counterparts that lacked it, but UW solution without PEN caused interstitial expansion equal to that of Euro-Collins solution with PEN. We conclude that macromolecules limit edema formation, but other constituents of UW solution limit edema formation also.

Acute hypoxia does not alter inter-alveolar perfusion distribution in unanesthetized rats

Effects of hypoxic vasoconstriction on inter-alveolar perfusion distribution (< or =1000 alveoli) have not been studied. To address this, we measured inter-alveolar perfusion distribution in the lungs of unanesthetized rats breathing 10% O(2). Perfusion distributions were measured by analyzing the trapping patterns of 4 microm diameter fluorescent latex particles infused into the pulmonary circulation. The trapping patterns were statistically quantified in confocal images of the dried lungs. Trapping patterns were measured in lung volumes that ranged between less than 1 and 1300 alveoli, and were expressed as the log of the dispersion index (logDI). A uniform (statistically random) perfusion distribution corresponds to a logDI value of zero. The more this value exceeds zero, the more the distribution is clustered (non-random). At the largest tissue volume (1300 alveoli) logDI reached a maximum value of 0.68+/-0.42 (mean+/-s.d.) in hypoxic rats (n = 6), 0.50+/-0.38 in hypercapnic rats (n.s.) and 0.48+/-0.25 in air-breathing controls (n.s.). Our results suggest that acute hypoxia did not cause significant changes in inter-alveolar perfusion distribution in unanesthetized, spontaneously breathing rats.

University of Wisconsin solution with butanedione monoxime and calcium improves rat lung preservation

BACKGROUNDnA limitation to fully using lung transplantation for patients with end-stage lung diseases is short, safe preservation time (4 to 6 hours). Our goal is to extend this to 24 hours or more, which would greatly improve clinical lung transplantation.nnnMETHODSnWe used the isolated perfused rat lung to test how two preservation solutions (low potassium dextran and University of Wisconsin solution) affected quality of lungs after 6, 12, and 24 hours of preservation. Also, we tested modifications of the University of Wisconsin solution, including reversing the ratio of Na/K, the addition of 1.5 mmol/L calcium, and the combination of calcium and butanedione monoxime, agents that improve cardiac preservation. After preservation at 4 degrees C, lungs were reperfused at 37 degrees C with a physiologically balanced solution. Pulmonary artery flow rate, airway peak inspiratory pressure, and tissue edema were used to assess degree of preservation and reperfusion injury.nnnRESULTSnLow potassium dextran solution gave poor preservation (decreased pulmonary artery flow, tissue edema) after 12 hours of cold storage. There were no differences between regular and reversed Na/K ratio University of Wisconsin solutions at 12 or 24 hours of preservation. Addition of calcium had no beneficial effect on lung preservation. However, University of Wisconsin solution with calcium and butanedione monoxime gave excellent 24-hour cold storage, with pulmonary artery flow rate, tissue edema, and airway peak inspiratory pressure equal to control (0 hours of preservation) lungs.nnnCONCLUSIONSnThe University of Wisconsin solution appears capable of lung preservation for up to 24 hours if modified to contain calcium and butanedione monoxime. The mechanism of action of butanedione monoxime may be related to the suppression of smooth muscle contraction resulting in vasodilation of the cold-stored lung on reperfusion.