Effects of prophylactic or therapeutic application of bovine haemoglobin HBOC-200 on ischaemia–reperfusion injury following acute coronary ligature in rats{dagger}

M. A. Burmeister*,{ddagger}, C. Rempf, T. G. Standl{ddagger}, S. Rehberg, S. Bartsch-Zwemke, T. Krause, S. Tuszynski, A. Gottschalk and J. Schulte am Esch

Department of Anaesthesia, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany

* Corresponding author. E-mail: burmeister{at}uke.uni-hamburg.de

Accepted for publication August 30, 2005.


    Abstract
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 Footnotes
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Background. Haemoglobin-based oxygen carriers (HBOCs) are assessed as blood substitutes in patients with perioperative anaemia including patients at risk for perioperative cardiac ischaemia. There is controversy as to whether HBOCs are beneficial or deleterious during ischaemia–reperfusion (I–R). Therefore the effects of HBOC-200 on I–R injury were evaluated in a randomized placebo-controlled animal trial.

Methods. Animals were randomized to receive either placebo i.v. without I–R (sham group, n=9), placebo i.v. with I–R (control group, n=10), HBOC-200 0.4 g kg–1 i.v. prior to I–R (prophylaxis group, n=12) or HBOC-200 0.4 g kg–1 i.v. during I–R (therapy group, n=15). I–R consisted of 25 min of acute ligature of the left coronary artery followed by 120 min of reperfusion. Measurements included assessment of the area at risk and infarct size using triphenyl tetrazolium chloride (TTC) stain, DNA single-strand breaks (in situ nick translation with autoradiography/densitometry) and cardiac arrhythmias.

Results. Infarct size within the area at risk was 62 (SD 15)% (control), 46 (10)% (prophylaxis, P<0.025 vs control) and 61 (9)% (therapy, P<0.85 vs control). The frequency of DNA single-strand breaks was reduced vs control in the sham (P<0.01) and prophylaxis (P<0.04) groups and was almost the same in the therapy group (P<0.75). The severity of cardiac arrhythmias during ischaemia was lower compared with control in the sham (P<0.001) and prophylaxis (P<0.039) groups, but there was no difference in the therapy group.

Conclusion. This study demonstrates that neither prophylactic nor therapeutic application of the cell-free haemoglobin solution HBOC-200 aggravates cardiac I–R injury. Furthermore, the prophylactic approach may offer a new opportunity for pretreatment of patients at risk for perioperative ischaemic cardiac events.

Keywords: blood, haemoglobin ; heart, arrhythmia ; heart, ischaemia ; model, rat ; special drugs


    Introduction
 Top
 Footnotes
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Haemoglobin based oxygen carriers (HBOCs) are assessed as blood substitutes in patients with perioperative anemia.1 In 2001 bovine HBOC-201 was the first drug approved in this context for routine use in South Africa. Several other formulations are currently under investigation. Unpromising results of clinical trials has led to the cancellation of several development projects in the past. In addition to the problem of demonstrating the clinical efficacy of HBOCs as a blood substitute, there are still concerns regarding side effects such as vasoconstriction which may explain why they have not yet been approved as blood substitutes in humans in Europe and the USA.

Patients with coronary artery disease undergoing non-cardiac surgery are at risk for perioperative ischaemia,2 3 and whether HBOC may be beneficial4 6 or deleterious7 8 with regard to myocardial ischaemia–reperfusion (I–R) injury is still under discussion. Deleterious effects may result from vasoconstriction or generation of an increased amount of reactive oxygen species. Since no data exist for bovine HBOC, the effects of HBOC-200, an analogue of the approved HBOC-201, on myocardial damage and I–R related cardiac arrhythmia have been evaluated in a prospective randomized blinded animal trial.

In addition to the well-established technique using triphenyl tetrazolium chloride (TTC) staining for the assessment of infarct size, we included a new autoradiographic technique of in situ nick translation for quantification of myocardial DNA single-strand breaks as an indicator of tissue damage.


    Methods
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 Footnotes
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Experiments were performed after approval had been obtained from the local committee for animal care (BAGS Hamburg) in accordance with the Guide for the Care and Use of Laboratory Animals.9

Anaesthesia was induced in male Sprague–Dawley rats (354 (SD 60) g) using S(+)-ketamine 80 mg kg–1 i.m. and midazolam 3 mg kg–1 i.m. The maximum volume was limited to 0.2 ml per site.10 Animals were placed on a heated rodent operating table (Harvard Apparatus, Boston, MA) and their lungs were insufflated with oxygen; they were monitored using three-lead ECG, pulse oximetry and a rectal temperature probe. After local infiltration with lidocaine 0.1% (0.5 ml) animals underwent tracheotomy, cannulation (2.0 mm) and mechanical ventilation (Inspira Advanced Safety Ventilator, Harvard Apparatus, Boston, MA). After insertion of a central venous catheter via the jugular vein anaesthesia was maintained intravenously using midazolam 10 mg kg–1 h–1 and fentanyl 7.5 µg kg–1 h–1. A continuous infusion of saline 0.9% (10–15 ml kg–1 h–1) was used to provide a central venous pressure of 8–12 mm Hg. All animals received an arterial line via the femoral artery with continuous measurement of the arterial blood pressure. End-expiratory carbon dioxide pressure () and arterial carbon dioxide pressure () were assessed (Capnostat, Marquette, Germany) during preparation and I–R.

Glass tubes of volume 150 µl (Clinitubes, Radiometer Medical, Copenhagen, Denmark) were used for repetitive blood sampling. Measurements included blood gases, Ca2+ and K+ concentrations (ABL 505, Radiometer Medical, Copenhagen, Denmark), and concentrations of total haemoglobin (Hbt), oxyhaemoglobin (HbO2), carboxyhaemoglobin (COHb), met-haemoglobin (MetHb) and plasma haemoglobin (Hbp) (OSM 3, Radiometer Medical, Copenhagen, Denmark). For measurement of Hbp 80 µl of arterial blood were centrifuged for 5 min at 5000 r.p.m. and the plasma was taken for analysis. The oxygen content () was measured directly (Lex-O2-ConTM, Lexington, MA). Blood samples were taken at baseline, at the end of the recovery period (recovery+30 min), after the first application of study solution or placebo (application 1+5 min), after occlusion of the coronary artery (occlusion +5 min), after the second application of study solution or placebo directly before reperfusion (occlusion +25 min) and twice during reperfusion (reperfusion +10 min/reperfusion +110 min).

After left-sided thoracotomy between the fourth and fifth costae a positive end-expiratory pressure of 5 cm H2O was applied. A ligature was placed around the left coronary artery according to a frequently used technique,11 and ischaemia was induced by tightening it. At the end of the preparation, after a recovery period of 30 min (time without stimulation following the period of instrumentation and surgical preparation of the animals), animals were randomized to one of four groups to receive placebo i.v. (2 ml NaCl) without I–R (sham, n=9), placebo i.v. with I–R (control, n=10), HBOC-200 0.4 g kg–1 i.v. 15 min before I–R (prophylaxis, n=12) or HBOC-200 0.4 g kg–1 i.v. during I–R (10 min after induction of ischaemia) (therapy, n=15). I–R consisted of 25 min of acute ligature of the left coronary artery followed by 120 min of reperfusion. The design is shown in Figure 1. Heart rate, mean arterial blood pressure, temperature and pulse oximetry were measured continuously during I–R. Arterial blood samples were taken repetitively for analysis of blood gases and concentrations of haemoglobin, electrolytes and glucose.



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Fig 1 Schematic overview of the study design starting with the period for recovery from preparation. Arrows indicate time of major measurements including blood sampling.

 
Study solution
OxyglobinTM (Biopure, Cambridge, MA) is a cell-free dark red haemoglobin solution in which bovine haemoglobin tetramers are glutaraldehyde polymerized, resulting in chains of 15 tetramers with an average molecular weight of 200 kDa. It has a p50 (oxygen affinity) of 36 mm Hg. The haemoglobin concentration is 12.0 (1.0) g dl–1, the colloid osmotic pressure is 17 mm Hg, pH is 7.6–7.9 and the osmolarity is 290–310 mOsm kg–1.

In order to detect the area at risk coronary ischaemia was induced for a second time after 120 min of reperfusion with the animals receiving 2 ml of blue dye intravenously (Patentblau, Sigma Chemicals, Germany).12 The animals were killed by injecting potassium chloride 2 ml. The heart was removed immediately, placed in a frozen (–20°C) heart matrix and cooled for 20 min at –20°C before being sliced into 1-mm sections. All slices were scanned from both sides and then every second slice was stained with triphenyl tetrazolium chloride (TTC). The remainder of the slices were embedded in tissue freezing medium (Jung, Leica Instruments, Germany) and stored at –80°C until required. TTC staining was performed according to published standards.13 When TTC staining was complete all slices were scanned from both sides to compensate for the missing information from the second half. This technique enabled us to perform the additional examination of the density of DNA single-strand breaks in parallel in the same heart.

The sizes of the left ventricle, the area at risk and the infarct were determined according to previously published standards13 14 using computer-based planimetry (ScionImageTM, Scion Corporation, Frederick, MD). All scanned areas were quantified independently by two blinded investigators. After double-checking for typing errors, the mean of the values was taken.

Assessment of DNA single-strand breaks
The in situ nick translation (ISNT) method15 was used to detect DNA single-strand breaks. DNA polymerase (E.coli DNA polymerase I, Kornberg Polymerase, Boehringer Mannheim, Germany) mediated template-dependent binding of the radioactive nucleotide desoxy-methyl-3H-thymidine 5'-triphosphate (Amersham Pharmacia Biotech, Amersham, UK) was performed at the 3'-OH ends of the broken strands.

A 1-mm slice from the middle of the heart (8 mm from the base) was used for the procedure. The slice was cut into 10-µm slices in a cooled ultramicrotome (CM 1800, Leitz, Nussloch, Germany) and these slices were placed in saline 0.9% prior to fixation in methanol. The dried slices were incubated with 700 µl ISNT kit at 21°C for 40 min. The reaction was stopped by addition of tetrasodium-pyrophosphate 1% (Sigma Chemicals, Germany). The positive control was produced by incubating slices from the sham group with DNase I prior to ISNT to induce a high number of DNA single-strand breaks. The negative control was produced by incubating slices from the sham group for ISNT without DNA polymerase.

Dried slices (five from each animal) were placed on paper and incubated on an X-ray film (Hyperfilm 3H, Amersham Pharmacia Biotech, Amersham, UK) in a cassette for 24 h. Five slices from four animals were included on each film, and five microscales and three slices from a negative and a positive control were added. The developed films were scanned followed by densitometry (Personal Densitometer SI/ImageQuantTM 4.1, Molecular Dynamics, Sunnyvale, CA). An example is shown in Figure 2B. All values are expressed as percentages of the mean microscale standard (relative density). Five areas of each slice were measured according to the scheme shown in Figure 2. The areas within the left ventricle included ischaemic and non-ischaemic tissue. Only three of the five slices for each animal were used for further calculations; the slices with the highest and lowest overall values for the left ventricle were excluded. Preparation of tissue and measurements were performed independently by blinded investigators.



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Fig 2 (A) Schematic view of a cross-section of the heart with defined areas for quantification of the density of DNA single-strand breaks: (1) left ventricle (overall) including (2) endocardial third, (3) middle third, (4) epicardial third and (5) area of the right ventricle. RV, lumen of right ventricle; LV, lumen of left ventricle). (B) Example of an autoradiograph of a myocardial transverse slice of one animal (control group). DNA single-strand breaks are more frequent in dark areas.

 
Arrhythmias
An ECG was recorded continuously from the limbs of all animals using electrodes for neonates (13953D, Agilent Technology, Andover, MA) and a monitor (Tram-200 A, Marquette, Milwaukee, WS). The ECG was printed at 25 mm s–1 (Direct Digital Writer Series 7100, Marquette, Milwaukee, WS). The cardiac arrhythmias were monitored and assessed in accordance with the Lambeth Conventions.16 The assessment was performed in a blinded manner using the original paper recordings. A validated score was used to quantify the severity of cardiac arrhythmias.17 The score consisted of six levels: 0, no ventricular extrasystoles (VES), ventricular tachycardia (VT), ventricular fibrillation (VF); 2, VES, one to five episodes of VT (more than four coupled VES); 3, more than five episodes of VT and/or one VF; 4, two to five episodes of VF; 5, more than five episodes of VF.

Statistics
Primary study endpoint
The study was designed to test the following hypothesis: the prophylactic or therapeutic application of the cell-free haemoglobin solution HBOC-200 reduces myocardial infarct size compared with a placebo-treated control group in a rat model of acute myocardial ischaemia–reperfusion.

Secondary study endpoints
In addition to the primary endpoint, effects on severity of cardiac arrhythmias and DNA single-strand breaks were assessed and compared with the control group.

Sample size calculation and statistical analysis
Based on the hypothesis for the primary study endpoint, a computer-assisted sample size calculation was performed (Instat, GraphPad, San Diego, CA, USA). We set a 65% infarct size within the area at risk as the predicted value for the control group. The smallest difference to be clinically significant was defined as 25%. A type I error of {alpha}=0.05 was permitted; with the alternative hypothesis, the null hypothesis would be retained with a type II error of ß=0.2.

The study was planned to include eight animals that completed the full study period per group. If an animal died after randomization but before completion of the 120 min reperfusion period the randomization number was used again. However, all randomized animals were included in the analysis of the frequency and severity of cardiac arrhythmias irrespective of survival time.

Computerized statistical analysis was performed using SPSS 9.0 (SPSS Inc.) and Instat 2.1 (GraphPad). Data are reported as mean (SD) unless otherwise indicated. Continuous variables were analysed using analysis of variance (ANOVA) with the two-tailed Dunnet t post hoc test. The control group was used as the reference. Ordinal data (e.g. arrhythmias) were compared using the Mann–Whitney U-test. A two-tailed P≤0.05 was regarded as significant.


    Results
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 Abstract
 Introduction
 Methods
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 Discussion
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Study population
A total of 46 animals were included in the study. In each of the four groups eight animals completed the whole study period. Fourteen animals died before the end of the 120 min reperfusion period (sham, one; control, two; therapy, seven; prophylaxis, four). In the therapy group five of the seven animals died before application of HBOC 200. No significant differences between groups were seen with respect to body weight.

Haemodynamics and body temperature
None of the measured baseline values differed between groups. All variables except mean arterial pressure (MAP) remained stable during the procedure. Application of HBOC-200 was followed by a 10–20 min increase in MAP in the prophylaxis group compared with control (P<0.05). A similar trend was also seen in the therapy group when HBOC-200 was given, but the increase was not significant (Table 1). During the rest of the observation time no significant differences were seen among groups.


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Table 1 Heart rate (HR), mean arterial blood pressure (MAP) and core temperature of all animals completing the whole study period before and during ischaemia–reperfusion. Data are mean (SD);

 
Haematology and blood gases
Baseline values did not differ. The plasma haemoglobin concentration increased in both the prophylaxis and the therapy groups. After application of HBOC-200 plasma haemoglobin concentration was elevated to a maximum of 1 g dl–1 followed by a moderate decrease over time in both the prophylaxis and the therapy groups (Table 2). Total haemoglobin concentration, oxygen content, and did not differ between groups during the whole observation period (Table 2).


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Table 2 Blood gases and haemoglobin concentrations of all animals completing the whole study period before and during ischaemia–reperfusion. Data are expressed as mean (SD).

 
Area at risk and infarct size
Infarct size in relation to the area at risk was significantly lower in the prophylaxis group than in the control group but no difference was seen after therapeutic application of HBOC-200 (Fig. 3). The size of the area at risk (percentage of left ventricle) was comparable between groups (control group, 30 (10)%; therapy group, 30 (10)%; prophylaxis group, 26 (7)%).



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Fig 3 Size of infarction within the area at risk. Data are expressed as mean (SD) percentage. n.a., not applicable.

 
DNA single-strand breaks
Quantification of DNA single-strand breaks (DNA-SSBs) within the left ventricle showed a lower density in the prophylaxis and sham group than in the control group irrespective of the region of assessment, but no effects were seen after therapeutic application. No overlaps of the 95% confidence intervals of the mean were seen between sham and control groups (Fig. 4AD).



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Fig 4 Relative density of DNA single-strand breaks in the myocardial tissue within groups: (A) left ventricle overall; (B) left ventricle epicardial third; (C) left ventricle middle; (D) left ventricle endocardial third; (E) area of the right ventricle. Data are expressed as mean with 95% confidence interval and extreme values (P=comparison vs control).

 
Cardiac arrhythmias
The severity of cardiac arrhythmias was lowest in the sham group and highest in the therapy and control groups. During ischaemia prophylactic application of HBOC-200 led to a significant reduction compared with control but no significant difference was seen in the overall assessment including ischaemia and reperfusion (Fig. 5).



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Fig 5 Severity of arrhythmias during (A) ischaemia and (B) ischaemia–reperfusion based on the scale proposed by Curtis and colleagues17 (0, No ventricular extrasystoles (VES), ventricular tachycardia (VT), ventricular fibrillation (VF); 2, VES, one to five episodes of VT (>4 coupled VES); 3, more than five episodes of VT and/or one VF; 4, two to five episodes of VF; 5, more than five episodes of VF). Data are given as frequencies of the highest scores for each animal in each group. *The event occurred in two animals before therapeutic application of HBOC-200.

 

    Discussion
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 Footnotes
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We have shown that prophylactic application of HBOC-200 0.4 g kg–1 before an acute coronary occlusion and reperfusion significantly reduces infarct size, density of DNA-SSBs and severity of cardiac arrhythmias compared with controls, but no beneficial effects were observed in the therapy group. Although HBOC-200 is for veterinary use only these findings are of high clinical relevance since HBOC-201, an analogue of HBOC-200 which differs only in the concentration of small haemoglobin molecules (molecular weight <30 kDa), is approved in South Africa for treatment of acute anaemia and has been used in clinical trials in Europe and the US. Although several patients with cardiovascular morbidities have been enrolled in phase III trials,18 no data regarding the effects of HBOC-201 or its analogue during acute cardiac ischaemia and reperfusion are available.

Some experimental trials have shown that cell-free oxygen carriers such as diaspirin cross-linked haemoglobin (DCLHb)4 19 and particularly perfluorocarbons20 can have beneficial effects on post-ischaemic tissue and improve the function of the ischaemic heart.2 Unfortunately, in most trials the drugs were applied during or after, but not before, ischaemia. This is important because blood substitutes are generally given during surgery, whereas severe cardiac ischaemia typically occurs in the early postoperative phase.2 3 In the clinical setting this could lead to a situation in which artificial oxygen carriers are within the circulation when an ischaemic cardiac event occurs.

In a recently reported trial, Caswell and colleagues22 administered HBOC-201 to dogs 30 min prior to 90 min of cardiac ischaemia followed by 270 min of reperfusion. Compared with a control group which was treated with saline 0.9% the infarct size within the area at risk could be reduced by 56%. These results are in accordance with our data, although they are more pronounced, which may be attributable to the higher dosage used. In addition, histological analysis revealed a significant reduction in neutrophil infiltration in the HBOC-201 group. The authors conclude that the positive effects of HBOC-201 are probably related to preloading the myocardium with oxygen or by aiding oxygen delivery to hypoxic tissue. In our trial we included two different administration time points, one before and one during ischaemia. Although it was not the intention of this study to investigate mechanisms of action of HBOC-200 during I–R in detail, the design gave us the opportunity to gain some insights into the time course of the underlying mechanisms. Since therapeutic application of HBOC-200 had no effect compared with control, the crucial effects must have already occurred during ischaemia and may be related to increased oxygen preload or delivery as noted above. In addition, at the time of the reperfusion some of the capillary bed is probably no longer open,23 restricting the effect of HBOC-200 to a lower part of the perfused microvasculature. The extent of this perfusion disturbance depends on the length of ischaemia, and this process goes on during reperfusion until its final size is reached, probably at 120 min as shown in a rabbit model.24 This microvascular occlusion is known to be a consequence of a complex interaction between endothelial cell damage, cell protrusion, vascular dysfunction, perivascular swelling and microembolisms produced by leucocytes and platelets and their interactions with the endothelium (‘sticking and rolling’). In this context HBOC-200 may become effective. This hypothesis is supported by the findings of Nolte and colleagues5 who examined the effects of DCLHb on microvascular integrity during I–R in a skin chamber model following haemorrhagic shock. These data demonstrate that DCLHb increases venular red blood cell velocity under both non-ischaemic and post-ischaemic conditions without inducing enhanced leucocyte–endothelium interaction in the microcirculation of striated skin muscle.5 The results of another study of pressure-induced I–R of striated skin muscle indicated that DCLHb reduced tissue damage in animals compared with Detranx-60 or isotonic saline, presumably through alterations in leucocyte–endothelial cell interactions.4 In a different setting of experimentally induced pancreatitis, systemic intravenous infusion of bovine haemoglobin (HBOC-200) significantly reduced microcirculatory dysfunction in the rat.25 In summary, the results of the studies noted above contribute to the hypothesis that haemoglobin solutions can attenuate I–R injury, at least in part, via a reduction of microvascular perfusion impairment.

The reduced severity of cardiac arrhythmias can be partially explained by the reduction of the size of infarction as described for perfluorocarbons,26 the alternative group of cell-free oxygen carriers. Nevertheless, since in our study the anti-arrhythmic effect was particularly noticeable during ischaemia, an additional mechanism appears to be reasonable and may be related to the presence of HBOC-200 in the area at risk at the moment of occlusion.

The only side-effect observed in this study, a moderate and temporary increase in MAP following the application of HBOC-200, can be explained by the well-known nitric oxide scavenging properties of haemoglobin solutions.1 It has been shown that the vasoconstrictive effect is also present in isolated rings of human arteries used for bypass surgery.27

The present study also demonstrates that densitometry of autoradiography following in situ nick translation allows good differentiation between the control and sham groups after 25 min ischaemia with 120 min of reperfusion. DNA-SSBs are known to be an early marker of critical cellular damage after oxidative stress (e.g. following transient ischaemia of the brain28) but this was not used for quantification of cellular damage in cardiac tissue. This technique offers additional information and can be used in parallel with conventional TTC stain as it requires only small amounts of tissue. One limitation is the inability to match the regions of the densitometry with the area of risk or the area of infarction assessed by TTC stain. Further studies should validate this technique for various lengths of ischaemia and reperfusion.

Limitations of the study
The present study was primarily designed to evaluate the effect of HBOC-200 on outcome variables, such as infarct size, DNA-SSB and arrhythmias, and not to clarify the underlying mechanisms. In addition, we used only one dose and cannot answer questions regarding dose dependency of the effects described. Moreover, we included only those animals in each group which had completed the 120 min of reperfusion in the analysis of infarct size and DNA-SSB. Since we had more premature deaths in the prophylaxis and therapy groups than in the control and the sham groups, we cannot exclude the possibility that the animals in the HBOC-treated groups represent a positive selection.

Furthermore, species differences always limit the conclusions which can be drawn based on an experimental animal trial. We have chosen a well-established model, the rat, which, with dog and rabbit, is the most frequently published experimental cardiac I–R model. Drugs such as ketamine can also affect infarct size. Since such effects have been demonstrated only for the racemic form,29 we used S(+)-ketamine in the present study.

Conclusion
Neither prophylactic nor therapeutic application of the cell-free haemoglobin solution HBOC-200 aggravated cardiac I–R injury. Furthermore, the prophylactic approach may offer a new opportunity for pretreatment of patients at risk for perioperative ischaemic cardiac events.


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 Footnotes
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
{dagger} The results were presented in part at the Congress of the European Society of Anaesthesia, Glasgow, UK, June 2003 (‘Best Abstract Award’), and at the Annual Meeting of the American Society of Anesthesiologists, San Francisco, CA, USA, October 2003. Back

{ddagger} Declaration of interest. T. G. Standl has received lecture honoraria and travel fees from Biopure Corporation, Boston, MA, the manufacturer of HBOC. The Department of Anaesthesiology, University Hospital, Hamburg-Eppendorf, received restricted grants from Biopure Corporation, Boston, MA, between 1994 and 1998 for animal and clinical phase II and III trials. M.A. Burmeister is Vice President Research and Development, Hospital Care Division, B. Braun Melsungen AG, Melsungen, Germany. B. Braun, a global health care supplier, cooperated with Biopure Corporation, Boston, MA, on HBOC development until 1996. The work presented in this paper was done independently of and without any support from B. Braun. Back


    References
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 Abstract
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 Methods
 Results
 Discussion
 References
 
1 Standl T. Haemoglobin-based erythrocyte transfusion substitutes. Expert Opin Biol Ther 2001; 1: 831–43[CrossRef][ISI][Medline]

2 Mangano DT, Wong MG, London MJ et al. Perioperative myocardial ischemia in patients undergoing noncardiac surgery–II: Incidence and severity during the 1st week after surgery. The Study of Perioperative Ischemia (SPI) Research Group. J Am Coll Cardiol 1991; 17: 851–7[Abstract]

3 Mangano DT, Browner WS, Hollenberg M et al. Association of perioperative myocardial ischemia with cardiac morbidity and mortality in men undergoing noncardiac surgery. The Study of Perioperative Ischemia Research Group. N Engl J Med 1990; 323: 1781–8[Abstract]

4 Pickelmann S, Nolte D, Leiderer R et al. Attenuation of postischemic reperfusion injury in striated skin muscle by diaspirin-cross-linked Hb. Am J Physiol 1998; 275: H361–8[ISI]

5 Nolte D, Botzlar A, Pickelmann S et al. Effects of diaspirin-cross-linked hemoglobin (DCLHb) on the microcirculation of striated skin muscle in the hamster: a study on safety and toxicity. J Lab Clin Med 1997; 130: 314–27[CrossRef][ISI][Medline]

6 Saetzler RK, Arfors KE, Tuma RF et al. Polynitroxylated hemoglobin-based oxygen carrier: inhibition of free radical-induced microcirculatory dysfunction. Free Radic Biol Med 1999; 27: 1–6[CrossRef][ISI][Medline]

7 Sadrzadeh S, Graf E, Panter S et al. Hemoglobin. A biologic fenton reagent. J Biol Chem 1984; 259: 14354–6[Abstract/Free Full Text]

8 Faassen A, Sundby S, Panter S et al. Hemoglobin: a lifesaver and an oxidant. how to tip the balance. Biomater Artif Cells Artif Organs 1988; 16: 93–104[ISI][Medline]

9 National Research Council IoLAR, Commission on Life Sciences. Guide for the Care and Use of Laboratory Animals. Washington, DC: National Academy Press, 1996

10 Waynforth H, Flecknell P. Experimental and Surgical Technique in the Rat, 2nd edn. San Diego, CA: Academic Press, 1992

11 Selye H, Bajusz E, Grasso S, Mendell P. Simple techniques for surgical occlusion of coronary vessels in the rat. Angiology 1960; 11: 398–407[Medline]

12 Ito WD, Schaarschmidt S, Klask R et al. Infarct size measurement by triphenyltetrazolium chloride staining versus in vivo injection of propidium iodide. J Mol Cell Cardiol 1997; 29: 2169–75[CrossRef][ISI][Medline]

13 Fishbein MC, Meerbaum S, Rit J et al. Early phase acute myocardial infarct size quantification: validation of the triphenyl tetrazolium chloride tissue enzyme staining technique. Am Heart J 1981; 101: 593–600[CrossRef][ISI][Medline]

14 Takashi E, Ashraf M. Pathologic assessment of myocardial cell necrosis and apoptosis after ischemia and reperfusion with molecular and morphological markers. J Mol Cell Cardiol 2000; 32: 209–24[CrossRef][ISI][Medline]

15 Rigby PW, Dieckmann M, Rhodes C, Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol 1977; 113: 237–51[CrossRef][ISI][Medline]

16 Walker MJ, Curtis MJ, Hearse DJ et al. The Lambeth Conventions: guidelines for the study of arrhythmias in ischaemia infarction, and reperfusion. Cardiovasc Res 1988; 22: 447–55[ISI][Medline]

17 Curtis MJ, Walker MJ. Quantification of arrhythmias using scoring systems: an examination of seven scores in an in vivo model of regional myocardial ischaemia. Cardiovasc Res 1988; 22: 656–65[ISI][Medline]

18 Levy JH, Goodnough LT, Greilich PE et al. Polymerized bovine hemoglobin solution as a replacement for allogeneic red blood cell transfusion after cardiac surgery: results of a randomized, double-blind trial. J Thorac Cardiovasc Surg 2002; 124: 35–42[Abstract/Free Full Text]

19 Habler O, Kleen M, Pape A et al. Diaspirin-crosslinked hemoglobin reduces mortality of severe hemorrhagic shock in pigs with critical coronary stenosis. Crit Care Med 2000; 28: 1889–98[CrossRef][ISI][Medline]

20 Rice HE, Virmani R, Hart CL et al. Dose-dependent reduction of myocardial infarct size with the perfluorochemical Fluosol-DA. Am Heart J 1990; 120: 1039–46[CrossRef][ISI][Medline]

21 McKenzie JE, Cost EA, Scandling DM et al. Effects of diaspirin crosslinked haemoglobin during coronary angioplasty in the swine. Cardiovasc Res 1994; 28: 1188–92[ISI][Medline]

22 Caswell JE, Strange MB, Rimmer DM 3rd et al. A novel hemoglobin-based blood substitute protects against myocardial reperfusion injury. Am J Physiol Heart Circ Physiol 2005; 288: H1796–801[Abstract/Free Full Text]

23 Reffelmann T, Hale SL, Li G, Kloner RA. Relationship between no reflow and infarct size as influenced by the duration of ischemia and reperfusion. Am J Physiol Heart Circ Physiol 2002; 282: H766–72[Abstract/Free Full Text]

24 Reffelmann T, Kloner RA. Microvascular reperfusion injury: rapid expansion of anatomic no reflow during reperfusion in the rabbit. Am J Physiol Heart Circ Physiol 2002; 283: H1099–107[Abstract/Free Full Text]

25 Strate T, Mann O, Kleinhans H et al. Systemic intravenous infusion of bovine hemoglobin significantly reduces microcirculatory dysfunction in experimentally induced pancreatitis in the rat. Ann Surg 2003; 238: 765–71[CrossRef][ISI][Medline]

26 Kolodgie FD, Dawson AK, Roden DM et al. Effect of Fluosol-DA on infarct morphology and vulnerability to ventricular arrhythmia. Am Heart J 1986; 112: 1192–201[CrossRef][ISI][Medline]

27 Ritchie AJ, Hartshorn S, Crosbie AE et al. The action of diaspirin cross-linked haemoglobin blood substitute on human arterial bypass conduits. Eur J Cardiothorac Surg 2000; 18: 241–5[Abstract/Free Full Text]

28 Chen J, Jin K, Chen M et al. Early detection of DNA strand breaks in the brain after transient focal ischemia: implications for the role of DNA damage in apoptosis and neuronal cell death. J Neurochem 1997; 69: 232–45[ISI][Medline]

29 Mullenheim J, Frassdorf J, Preckel B et al. Ketamine, but not S(+)-ketamine, blocks ischemic preconditioning in rabbit hearts in vivo. Anesthesiology 2001; 94: 630–6[CrossRef][ISI][Medline]





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