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.
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Abstract |
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Methods. Animals were randomized to receive either placebo i.v. without IR (sham group, n=9), placebo i.v. with IR (control group, n=10), HBOC-200 0.4 g kg1 i.v. prior to IR (prophylaxis group, n=12) or HBOC-200 0.4 g kg1 i.v. during IR (therapy group, n=15). IR 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 IR 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
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Introduction |
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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 ischaemiareperfusion (IR) 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 IR 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.
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Methods |
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Anaesthesia was induced in male SpragueDawley rats (354 (SD 60) g) using S(+)-ketamine 80 mg kg1 i.m. and midazolam 3 mg kg1 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 kg1 h1 and fentanyl 7.5 µg kg1 h1. A continuous infusion of saline 0.9% (1015 ml kg1 h1) was used to provide a central venous pressure of 812 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 IR.
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 IR (sham, n=9), placebo i.v. with IR (control, n=10), HBOC-200 0.4 g kg1 i.v. 15 min before IR (prophylaxis, n=12) or HBOC-200 0.4 g kg1 i.v. during IR (10 min after induction of ischaemia) (therapy, n=15). IR 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 IR. Arterial blood samples were taken repetitively for analysis of blood gases and concentrations of haemoglobin, electrolytes and glucose.
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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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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 ischaemiareperfusion.
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 =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 MannWhitney U-test. A two-tailed P0.05 was regarded as significant.
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Results |
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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 1020 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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Discussion |
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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 IR 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 IR 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 leucocyteendothelium interaction in the microcirculation of striated skin muscle.5 The results of another study of pressure-induced IR of striated skin muscle indicated that DCLHb reduced tissue damage in animals compared with Detranx-60 or isotonic saline, presumably through alterations in leucocyteendothelial 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 IR 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 IR 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 IR 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 |
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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.
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