C. Rempf

Improvement of Impaired Microcirculation and Tissue Oxygenation by Hemodilution with Hydroxyethyl Starch plus Cell-Free Hemoglobin in Acute Porcine Pancreatitis

Aims: To avoid the progression from mild edematous acute pancreatitis (AP) to the severe necrotizing form, one therapeutic option is to improve pancreatic microcirculation and tissue oxygenation. The aim of the study was to evaluate the influence of improved rheology (isovolemic hemodilution) plus enhanced oxygen supply (bovine hemoglobin HBOC-301) on pancreatic microcirculation, tissue oxygenation and survival in severe acute experimental pancreatitis. Methods: Severe AP was induced in 39 pigs (25–30 kg BW) by stimulation with intravenous administration of cerulein plus a pressure- and volume-controlled 10-min intraductal infusion of glycodeoxycholic acid. Seventy-five minutes after induction of AP, animals were randomized and hemodiluted isovolemically (PAOP constant) with either 10% hydroxyethyl starch (HES) 200,000/0.5 plus HBOC-301 (+0.6 g/dl plasmatic hemoglobin; Oxyglobin®, Biopure, Cambridge, Mass., USA), or 10% HES 200,000/0.5, or Ringer’s solution to a hematocrit of 15%. Hemodynamics, oxygen transport parameters, pancreatic microcirculation and tissue oxygen tension were evaluated over 6 h. Then the abdomen was closed, animals were extubated and observed for 6 days. After that, the surviving animals were sacrificed and specimens were taken from the pancreas. The histopathologic findings were scored by two blinded pathologists who quantified acinar necrosis, fat necrosis, inflammation and edema. Results: Isovolemic hemodilution with HES plus HBOC-301 reduced mortality and preserved pancreatic microcirculation compared with Ringer’s solution, but was not significantly different from hemodilution with HES alone. Only treatment with HES plus HBOC-301 normalized pancreatic tissue oxygen tension compared with IHD with HES or Ringer’s solution alone. Conclusions: IHD with HES plus HBOC-301 as a combination of rheologic and O2-delivering therapy may represent a novel therapeutic option for treatment of AP.

Administration of bovine polymerized haemoglobin before and during coronary occlusion reduces infarct size in rabbits

BACKGROUND Haemoglobin-based oxygen carriers (HBOC) seem to increase the risk of mortality and myocardial infarction in clinical trials. Therefore, we designed this randomized placebo-controlled animal study to evaluate the effects of prophylactic and therapeutic administration of HBOC in a myocardial ischaemia-reperfusion model with respect to infarct size and areas of impaired perfusion (no reflow, NR). METHODS Thirty-two anaesthetized, mechanically ventilated rabbits were randomized to one of the four groups. Group G1 received 0.4 g kg(-1) i.v. HBOC-200 25 min before coronary artery occlusion, G2 received the same dose i.v. 10 min after occlusion, and G3 and 4 received i.v. saline. G1, 2, and 3 were subjected to 30 min occlusion of left coronary artery followed by 240 min of reperfusion. G4 was treated without ischaemia-reperfusion. Measurement included assessment of the area at risk and infarct size using triphenyltetrazolium chloride stain and areas of NR using thioflavin stain. Ischaemia-reperfusion was confirmed by microspheres technique. RESULTS Infarct size as a percentage of the area at risk was significantly reduced in G1 [25 (sd 13)%, P=0.026] and G2 [22 (20)%, P=0.009] compared with G3 [48 (17)%]. The areas of NR in percentage of the area at risk [G1, 26 (15)%; G2, 34 (22)%; G3, 36 (12)%; G4, 5 (3)%] did not differ between the groups of animals undergoing coronary occlusion and reperfusion. CONCLUSIONS Prophylactic and therapeutic administration of HBOC-200 reduces infarct size in myocardial ischaemia and reperfusion in rabbits. This reduction of infarct size is not accompanied by an improvement of areas of NR.

Enhanced central organ oxygenation after application of bovine cell-free hemoglobin HBOC-201.

Objectif Les effets de ľanemie par hemodilution ou hemodilution isovolemique (HDI) sur ľextraction ďoxygene et ľoxygenation tissulaire dans les organes peripheriques apres ľutilisation de transporteur ďoxygene a base ďhemoglobine comme HBOC201 ont ete tres etudies, mais on connait peu les proprietes de ľoxygenation tissulaire des solutions ďhemoglobine dans les organes centraux comme le foie.PurposeWhile the effects of dilutional anemia or isovolemic hemodilution (IHD) on the oxygen extraction and tissue oxygenation in peripheral organs after application of hemoglobin-based oxygen carriers like HBOC-201 have been studied intensively, little is known about tissue oxygenation properties of hemoglobin solutions in central organs like the liver.MethodsTwelve Foxhounds were anesthetized and then randomized to either a control group without hemodilution (Group 1) or underwent first step isovolemic hemodilution (pulmonary artery occlusion pressure constant) with Ringer’s solution (Group 2) to a hematocrit of 25% with second step infusion of HBOC-201 until a hemoglobin concentration of +0.6 g·dL-1 was reached. Tissue oxygen tensions (tpO2) were measured in the gastrocnemius muscle using a polarographic needle probe, and in the liver using a flexible polarographic electrode.ResultsWhile arterial oxygen content and oxygen delivery decreased with hemodilution in Group 2, global liver and muscle oxygen extraction ratio increased after hemodilution and additional application of HBOC-201. Hemodilution and application of HBOC-201 provided augmentation of the mean liver tpO2 (baseline: 48 ± 9, 20 min: 53 ± 10, 60 min: 67 ± 11*, 100 min: 68 ± 7*; *P < 0.05vs baseline and Group 1), while oxygen tensions in Group 1 remained unchanged. Oxygen tension in the skeletal muscle increased after hemodilution and additionally after application of HBOC-201 in comparison to baseline and to the control group (P < 0.05).ConclusionIn the present animal model, IHD with Ringer’s solution and additional application of HBOC-201 increased oxygen extraction and tpO2 in the liver and skeletal muscle, in parallel and in comparison with baseline values and a control group.RésuméObjectifLes effets de ľanémie par hémodilution ou hémodilution isovolémique (HDI) sur ľextraction ďoxygène et ľoxygénation tissulaire dans les organes périphériques après ľutilisation de transporteur ďoxygène à base ďhémoglobine comme HBOC201 ont été très étudiés, mais on connaît peu les propriétés de ľoxygénation tissulaire des solutions ďhémoglobine dans les organes centraux comme le foie.MéthodeDouze Fox-hounds ont été anesthésiés, puis assignés à un groupe témoin sans hémodilution (Groupe 1) ou soumis à la première étape de ľhémodilution isovolémique (pression artérielle pulmonaire bloquée constante) avec une solution de Ringer (Groupe 2) jusqu’à un hématocrite de 25 % et ont reçu une perfusion de seconde étape avec HBOC-201 pour obtenir une concentration ďhémoglobine de +0,6 g·dL-1 . Les tensions en oxygène tissulaire (tpO2) ont été mesurées dans le muscle gastrocnémien en utilisant une sonde à injection polarographique et dans le foie au moyen ďune électrode polarographique flexible.RésultatsTandis que le contenu en oxygène artériel et la distribution ďoxygène ont diminué avec ľhémodilution dans le Groupe 2, le ratio ďextraction globale ďoxygène hépatique et musculaire a augmenté après ľhémodilution et ľusage additionnel de HBOC-201. ľhémodilution et HBOC-201 ont fait augmenter la tpO2 hépatique moyenne (mesures de base : 48 ± 9, 20 min : 53 ± 10, 60 min : 67 ± 11*, 100 min : 68 ± 7*; *P < 0,05 vs mesures de base et Groupe 1), tandis que les tensions en oxygène sont demeurées inchangées dans le Groupe 1. La tension en oxygène dans le muscle squelettique a augmenté après ľhémodilution et aussi après ľusage de HBOC-201 en comparaison des mesures de base et du groupe témoin. (P < 0,05).ConclusionChez le modèle animal utilisé, ľHDI avec une solution de Ringer et ľusage additionnel de HBOC-201 ont augmenté ľextraction ďoxygène et la tpO2 dans le foie et le muscle squelettique, parallèlement et comparativement aux valeurs de base et au groupe témoin.

[Amélioration de ľoxygénation des organes centraux après ľusage ďhémoglobine bovine acellulaire HBOC-201]

Objectif Les effets de ľanemie par hemodilution ou hemodilution isovolemique (HDI) sur ľextraction ďoxygene et ľoxygenation tissulaire dans les organes peripheriques apres ľutilisation de transporteur ďoxygene a base ďhemoglobine comme HBOC201 ont ete tres etudies, mais on connait peu les proprietes de ľoxygenation tissulaire des solutions ďhemoglobine dans les organes centraux comme le foie.PurposeWhile the effects of dilutional anemia or isovolemic hemodilution (IHD) on the oxygen extraction and tissue oxygenation in peripheral organs after application of hemoglobin-based oxygen carriers like HBOC-201 have been studied intensively, little is known about tissue oxygenation properties of hemoglobin solutions in central organs like the liver.MethodsTwelve Foxhounds were anesthetized and then randomized to either a control group without hemodilution (Group 1) or underwent first step isovolemic hemodilution (pulmonary artery occlusion pressure constant) with Ringer’s solution (Group 2) to a hematocrit of 25% with second step infusion of HBOC-201 until a hemoglobin concentration of +0.6 g·dL-1 was reached. Tissue oxygen tensions (tpO2) were measured in the gastrocnemius muscle using a polarographic needle probe, and in the liver using a flexible polarographic electrode.ResultsWhile arterial oxygen content and oxygen delivery decreased with hemodilution in Group 2, global liver and muscle oxygen extraction ratio increased after hemodilution and additional application of HBOC-201. Hemodilution and application of HBOC-201 provided augmentation of the mean liver tpO2 (baseline: 48 ± 9, 20 min: 53 ± 10, 60 min: 67 ± 11*, 100 min: 68 ± 7*; *P < 0.05vs baseline and Group 1), while oxygen tensions in Group 1 remained unchanged. Oxygen tension in the skeletal muscle increased after hemodilution and additionally after application of HBOC-201 in comparison to baseline and to the control group (P < 0.05).ConclusionIn the present animal model, IHD with Ringer’s solution and additional application of HBOC-201 increased oxygen extraction and tpO2 in the liver and skeletal muscle, in parallel and in comparison with baseline values and a control group.RésuméObjectifLes effets de ľanémie par hémodilution ou hémodilution isovolémique (HDI) sur ľextraction ďoxygène et ľoxygénation tissulaire dans les organes périphériques après ľutilisation de transporteur ďoxygène à base ďhémoglobine comme HBOC201 ont été très étudiés, mais on connaît peu les propriétés de ľoxygénation tissulaire des solutions ďhémoglobine dans les organes centraux comme le foie.MéthodeDouze Fox-hounds ont été anesthésiés, puis assignés à un groupe témoin sans hémodilution (Groupe 1) ou soumis à la première étape de ľhémodilution isovolémique (pression artérielle pulmonaire bloquée constante) avec une solution de Ringer (Groupe 2) jusqu’à un hématocrite de 25 % et ont reçu une perfusion de seconde étape avec HBOC-201 pour obtenir une concentration ďhémoglobine de +0,6 g·dL-1 . Les tensions en oxygène tissulaire (tpO2) ont été mesurées dans le muscle gastrocnémien en utilisant une sonde à injection polarographique et dans le foie au moyen ďune électrode polarographique flexible.RésultatsTandis que le contenu en oxygène artériel et la distribution ďoxygène ont diminué avec ľhémodilution dans le Groupe 2, le ratio ďextraction globale ďoxygène hépatique et musculaire a augmenté après ľhémodilution et ľusage additionnel de HBOC-201. ľhémodilution et HBOC-201 ont fait augmenter la tpO2 hépatique moyenne (mesures de base : 48 ± 9, 20 min : 53 ± 10, 60 min : 67 ± 11*, 100 min : 68 ± 7*; *P < 0,05 vs mesures de base et Groupe 1), tandis que les tensions en oxygène sont demeurées inchangées dans le Groupe 1. La tension en oxygène dans le muscle squelettique a augmenté après ľhémodilution et aussi après ľusage de HBOC-201 en comparaison des mesures de base et du groupe témoin. (P < 0,05).ConclusionChez le modèle animal utilisé, ľHDI avec une solution de Ringer et ľusage additionnel de HBOC-201 ont augmenté ľextraction ďoxygène et la tpO2 dans le foie et le muscle squelettique, parallèlement et comparativement aux valeurs de base et au groupe témoin.

Influence of the Hemoglobin Solution HBOC-201 on Tissue Oxygenation in the Rat R1H-Tumor

Background: HBOC-201 is an ultra purified bovine hemoglobin solution. It has already been used in clinical phase II/III trials for emergency treatments. Animal experiments have shown that HBOC-201 is highly effective in tissue oxygenation. The study was performed in order to assess the potential of low dose HBOC-201 to improve tumor oxygenation. Methods: 30 rats with a subcutaneously growing rhabdomyosarcoma R1H tumor were randomly assigned either to be ventilated with carbogen (n = 10), or to receive an IV injection of 0.3 g/kg HBOC-201 (n = 10) or a combination of 0.3 g/kg HBOC-201 and carbogen breathing (n = 10). Under general anesthesia the effects of the respective treatment on the tissue oxygen tension (tpO2) of the tumor were determined using a flexible stationary probe at baseline (b) and 15 and 60 min after application of the respective medication. Results: HBOC-201 alone failed to improve tumor tpO2 (b: 1.3 ± 1.2 mmHg; 15 min: 1.4 ± 1 mmHg; 60 min: 1 ± 1 mmHg). In combination with carbogen the mean tpO2 of the tumor raised in comparison to baseline values (b: 3.1 ± 4.6 mmHg; 15 min: 8.5 ± 11* mmHg; 60 min: 4.8 ± 5 mmHg; *p < 0.05 vs. b), but this effect was less pronounced than the increase in tpO2 by carbogen alone (b: 3.4 ± 3.4 mmHg; 15 min: 9 ± 10* mmHg; 60 min: 13 ± 19* mmHg; *p < 0.05 vs. b). Conclusion: The application of low dose hemoglobin solution HBOC-201 does not result in improvement of tissue oxygenation in the rat rhabdomyosarcoma R1H.

Effects of a prophylactic or therapeutic application of perflubron emulsion on myocardial ischaemia-reperfusion injury in rats.

Background and objective: The efficacy of administering a perfluorochemical‐based oxygen therapeutic such as perflubron emulsion (OxygentTM) prior to ischaemia is currently unknown, although there is evidence for potential beneficial effects for the perioperative treatment in cardiac risk patients. This experimental study investigated the efficacy of perflubron emulsion in preventing reperfusion injury and myocardial infarction size after coronary ischaemia and reperfusion. The perflubron emulsion was given either in a prophylactic manner, prior to induction of myocardial ischaemia, or as a therapeutic agent given during ischaemia. Methods: Thirty‐two anaesthetized and mechanically ventilated rats were subjected to 25 min occlusion of the left coronary artery followed by 120 min reperfusion. Animals were randomized to one of four groups: Group 1 was treated with administration of 6 g kg−1 intravenous perflubron emulsion 25 min before occlusion; Group 2 received the same dose 10 min after occlusion; and Groups 3 and 4 received no perflubron emulsion. Inspired O2 (FiO2) concentration was maintained at 1.0 in Groups 1, 2 and 3 and at 0.35 in Group 4. Results: Neither prophylactic nor therapeutic perflubron emulsion treatment reduced infarct size measurements by triphenyltetrazolium‐chloride staining or severity of cardiac arrhythmias in comparison to the hyperoxic control group. However, prophylactic application of perflubron emulsion reduced areas of impaired perfusion vs. Group 3 assessed by in vivo staining with Thioflavin‐S while no significant effect was seen in Groups 2 and 4 vs. 3. Density of DNA single‐strand breaks in the ventricle was increased in all groups ventilated with 100% oxygen. Conclusion: Although administration of perflubron emulsion did not reduce infarct size, areas of impaired perfusion were significantly mitigated when perflubron emulsion was administered prior to coronary occlusion. However, a high oxygen concentration may provoke DNA strand breaks during reperfusion after ischaemia. Further studies must clarify whether enhanced oxidative stress outweighs the advantage of improved areas of impaired perfusion following perflubron emulsion.

Effects of isovolaemic haemodilution on oxygenation of liver and skeletal muscle.

Background and objective: Hydroxyethyl starch is frequently used for volume substitution during surgical procedures and for isovolaemic haemodilution. Haemodilution has also been shown to improve tissue oxygen tension in skeletal muscle. However, effects of this volume substitute on tissue oxygen tension of the liver during haemodilution remains unknown. Methods: Fourteen foxhounds were anaesthetized with fentanyl/midazolam and mechanically ventilated with 30% oxygen. Following splenectomy animals were randomly assigned to a control group without haemodilution but fluid substitution with Ringers lactate (Group C) or underwent isovolaemic haemodilution to a haematocrit of 25% with hydroxyethyl starch 70/0.5 (Group H). Haemodynamic parameters and oxygen transport during 100 min following isovolaemic haemodilution were measured. Liver oxygen tension was recorded using a flexible polarographic electrode tonometer, whereas in the muscle a polarographic needle probe was used. Results: Animal characteristics and baseline haematocrit were similar in both groups. At baseline the tissue oxygen tension of liver and skeletal muscle were not different between groups. Haemodilution with hydroxyethyl starch 70/0.5 provided augmentation of mean liver tissue oxygen tension (baseline: 46 ± 13 mmHg; 20 min: 60.3 ± 12 mmHg*; 60 min: 60 ± 16 mmHg*; 100 min: 63 ± 16 mmHg*; *P < 0.05 vs. baseline), while oxygen tensions in Group C remained unchanged (baseline: 48 ± 16 mmHg; 20 min: 52 ± 19 mmHg; 60 min: 49 ± 12 mmHg; 100 min: 52 ± 16 mmHg) and no differences could be detected between groups. Oxygen tension in skeletal muscle changed as follows: Group H ‐ baseline: 24 ± 32 mmHg; 20 min: 32 ± 3 mmHg*†; 60 min: 33 ± 7 mmHg*†; 100 min: 33 ± 11 mmHg†. Group C ‐ baseline: 22 ± 6 mmHg; 20 min: 21 ± 3 mmHg; 60 min: 24 ± 4 mmHg; 100 min: 18 ± 4 mmHg (*P < 0.05 vs. baseline, †P < 0.05 vs. Group C). Conclusion: In this animal model, isovolaemic haemodilution with hydroxyethyl starch 70/0.5 increased tissue oxygen tension in liver and skeletal muscle in comparison with baseline values. However, when compared between groups haemodilution only resulted in an increase of tissue oxygen tension in the muscle but not in the liver.

Failed Induction of Heme Oxygenase 1 in Endothelial Cells Exposed to the Hemoglobin Based Oxygen Carrier Oxyglobin

We investigated the effect of the bovine hemoglobin based oxygen carrier HBOC-200 (Oxyglobin®) in rat aortic endothelial cells (RAEC) on the activation of heme oxygenase 1 (HO-1). RAEC were incubated in the presence of 75 μ M (G1) or 225 μ M (G2) HBOC-200. The positive control (G3) was performed by incubation with 50 μ M Hemin. For negative control (G4) cells were incubated with medium alone. G2 and G3 reached a significant increase of bilirubin concentration compared to G4. A positive HO-1 signal in the Western Blot was seen in G3 12 and 24 h after incubation. The Western Blot of G1, G2 and G4 showed no HO-1 signal. These data suggest that HBOC-200 in the applied dosage cannot induce HO-1 expression in RAEC, and may be degraded by isoenzymes at a lower level.