- Email: [email protected]
Administration of bovine polymerized haemoglobin before and during coronary occlusion reduces infarct size in rabbits‡
British Journal of Anaesthesia 103 (4): 496–504 (2009)
doi:10.1093/bja/aep233
Advance Access publication August 26, 2009
Administration of bovine polymerized haemoglobin before and during coronary occlusion reduces infarct size in rabbits‡ C. Rempf1, T. Standl2, K. Schenke3, K. Chammas4, A. Gottschalk5, M.-A. Burmeister6† and A. Gottschalk7* 1
*Corresponding author. E-mail: [email protected] 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 kg21 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)%, P¼0.026] and G2 [22 (20)%, P¼0.009] 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. Br J Anaesth 2009; 103: 496–504 Keywords: heart, coronary occlusion; heart, ischaemia; model, rabbit Accepted for publication: July 7, 2009
Cell-free haemoglobin-based oxygen carriers (HBOC) were primarily developed for replacement of red blood cells. The haemoglobin-glutamer 200 (HBOC-200; Oxyglobinw) is a glutaraldehyde polymerized bovine haemoglobin. Glutaraldehyde works as a cross-linking agent, which stabilizes the tetramer form of haemoglobin by non-specific modifications to both a- and b-globin chains and then as a protein surface polymerizing agent that produces a polymer.1 2
In contrast to the veterinary product Oxyglobinw (HBOC-200), the solution Hemopurew (HBOC-201) is approved for sale in South Africa for the treatment of †
Declaration of interest. M.-A.B. is employed by B. Braun Melsungen AG, Melsungen, Germany, an international Medical Device and Pharmaceutical Manufacturer. ‡ This article is accompanied by Editorial III.
# The Author [2009]. Published by Oxford University Press on behalf of the British Journal of Anaesthesia. All rights reserved. For Permissions, please email: [email protected]
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Department of Anaesthesiology, University Medical Center Schleswig-Holstein, Campus Lu¨beck, Germany. 2 Department of Anaesthesiology and Critical Care Medicine, Academic Hospital Solingen, Germany. 3 Department of Cardiology and Pneumology, Altona General Hospital, Hamburg, Germany. 4Department of Radiology, Children’s Hospital Altona, Hamburg, Germany. 5Department of Anaesthesiology and Intensive Care Medicine, University Hospital Muenster, Germany. 6Department of Anaesthesiology, University Hospital Hamburg Eppendorf, Germany. 7Department of Anaesthesiology, Intensive Care Medicine and Pain Therapy, Knappschaftskrankenhaus Bochum Langendreer, University Hospital Bochum, Germany
Haemoglobin solution reduces infarct size
Methods This study was approved by the local committee for animal care (Behoerde fu¨r Arbeit, Gesundheit und Soziales,
Hamburg, Germany) in accordance with the Guide for the Care and Use of Laboratory Animals.8
Experimental protocol The study protocol (Fig. 1) was designed to assess the influence of prophylactic and therapeutic administration of HBOC-200 on infarct size and areas of impaired perfusion after 30 min of coronary artery occlusion (CAO) and 240 min of reperfusion in an animal model of low coronary collateralization. The rabbits were randomized to one of the four groups. Animals received either 0.4 g kg21 HBOC-200 25 min before ischaemia (G1 prophylaxis, n¼8) or 0.4 g kg21 HBOC-200 10 min after ischaemia (G2 therapy, n¼8). Control groups obtained the equivalent volume of saline with ischaemia and reperfusion for positive controls (G3, n¼8) and without ischaemia and reperfusion for negative controls (G4, n¼8). All fluids were infused via the ear vein over a 5 min period.
Study solution HBOC-200 (Oxyglobinw, Biopure, Cambridge, MA, USA) is based on glutaraldehyde-polymerized, ultrapurified bovine haemoglobin formulated in a modified lactated Ringer’s solution. It contains 13.0 g dl21 of cell-free haemoglobin with a p50 (oxygen affinity) of 36 mm Hg. The oncotic pressure of HBOC-200 is similar to 5% albumin, and the osmolality is similar to normal plasma. HBOC-200 used in our study was bought from a veterinary pharmacy. HBOC bags were stored in a special box as HBOC rapidly oxidizes to met-haemoglobin with consequences
Group 1: prophylaxis: n=8 HBOC Baseline
NaCl Ischaemia 30 min
Reperfusion 240 min
HBOC Ischaemia 30 min
Reperfusion 240 min
NaCl Ischaemia 30 min
Reperfusion 240 min
Group 2: therapy: n=8 NaCl Baseline Group 3: positive control: n=8 NaCl Baseline Group 4: negative control: n=8 NaCl
NaCl
Baseline time (h:mm) 0:00 0:05 0:10 0:15 0:20 0:25 0:30 0:35 0:40 0:45 0:50 0:55 1:00 1:05 1:10 1:12
BGA
BGA
BGA
BGA RMBF
BGA RMBF
Fig 1 Schematic overview of study design. BGA, blood gas analysis; RMBF, regional myocardial blood flow.
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5:20
BGA Thioflavin Patent blue Infarct size
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acutely anaemic surgical patients. Both solutions are similar with the exception of absolute molecular weight distribution of the polymers. It seems that haemoglobin in HBOC may be transported in blood plasma to areas where red blood cell flow is not adequate. Further, enhanced oxygen off-loading through capillaries might be beneficial to patients with cardiovascular risks.3 4 However, these artificial oxygen carriers increase mean arterial pressure (MAP) and pulmonary artery pressure and also systemic and pulmonary vascular resistance.5 6 This property of cell-free haemoglobin solutions might be harmful in patients with coronary heart disease. A recent meta-analysis including all available clinical HBOC trials found an increased risk of mortality and myocardial infarction.7 HBOC-induced vasoconstriction may increase areas of impaired perfusion, so-called ‘no reflow’ (NR) phenomenon, which may compromise tissue oxygen supply. The NR phenomenon is observed after transient coronary occlusion and characterized by areas of hypoperfusion and decreased return of regional myocardial blood flow (RMBF). Since only very limited data exist, we performed a prospective, randomized, and blinded animal trial to evaluate the effects of HBOC-200 given in a prophylactic and therapeutic manner on myocardial infarct size and ischaemia/reperfusion-related areas of impaired perfusion. We hypothesized that the administration of HBOC results in a decrease in infarct size and areas of impaired perfusion.
Rempf et al.
on oxygen binding and coagulation parameters.9 After closure of the box, air was evacuated and simultaneously the box was filled with nitrogen for 5 min. Safe closure of the box was confirmed using a negative pressure within the box. Before using the HBOC in the next animal, met-haemoglobin was measured. If met-haemoglobin was .5%, the solution was not accepted and a new bag was opened.
Surgical preparation
Haemodynamics During the whole experiment, heart rate, MAP and systolic arterial pressure, temperature, and pulse oximetry were continuously measured and recorded (Marquette Tramscope 12, Milwaukee, WI, USA). To assess the influence of haemodynamic parameters on myocardial oxygen consumption, we calculated the rate pressure product
RPP ¼
ðheart rateÞ ðarterial systolic blood pressureÞ 1000
Blood gas analysis Arterial blood samples were obtained repetitively in 150 ml glass tubes (Clinitubes, Radiometer Medical, Kopenhagen, Denmark). Measurements included blood gases, electrolytes, and glucose (ABL 505, Radiometer Medical, Copenhagen, Denmark), and concentrations of total haemoglobin (Hb), oxyhaemoglobin (HbO2), carboxyhaemoglobin (COHb), met-haemoglobin (MetHb), and plasmatic haemoglobin (Hbp) (OSM 3, Radiometer Medical, Copenhagen, Denmark). For measurement of plasmatic haemoglobin, 80 ml of arterial blood was centrifuged for 5 min at 2240 g and the plasma was obtained for analysis. Blood samples were obtained at baseline (5 min before prophylactic treatment), 10 min after prophylactic treatment, 5 and 20 min during ischaemia, and 2 and 240 min after reperfusion (Fig. 1).
Measurement of area at risk, impaired perfusion, and infarction At the end of the protocol, the area of impaired perfusion was delineated by an injection of the fluorescent dye thioflavin-S (1 ml kg21, Sigma Chemicals, Munich, Germany) via the left atrial catheter. The dye was prepared as a 4% solution in a warm saline solution, centrifuged for 10 min at 230 g and subsequently filtered to remove particles (pore size 0.2 mm) and injected manually over 20 s. Immediately after injection of thioflavin-S, the coronary artery was re-occluded followed by an injection of a blue dye (Patentblau 1 ml kg21, Sigma Chemicals) through the left atrial catheter to define the area at risk. After 1 min, when the perfused areas of the heart and the lungs were stained blue, the deeply anaesthetized animals were killed by i.v. injection of potassium chloride 7.45% (2 mmol kg21). The heart was rapidly excised and rinsed in cold isotonic saline, placed in a precooled (2208C) heart matrix for rabbits (ASI Instruments, Warren, MI, USA) and frozen for 20 min at 2208C before slicing into 2 mm transverse rings from the apex to the base. The apex of the heart was returned for measurements of RMBF in the area at risk. The remaining slices were first scanned to identify the ‘area at risk’ (uncoloured by the blue dye) and then photographed in the dark under ultraviolet light using a Fluor-STM Multiimager (Bio-Rad, Hercules, CA, USA) to determine areas, which were not perfused by thioflavin-S. Thioflavin-S is a fluorescent vital dye staining the endothelium of the blood vessels,11 and thus the normally perfused myocardium (fluorescent) was easily differentiated from the non-perfused (non-fluorescent) myocardium. Subsequently, slices were incubated for 10 min at 378C in a 1% solution of
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Male New Zealand white rabbits (weight, 3.0 kg) were anaesthetized by i.m. injection of midazolam (1.5 mg kg21) and S-ketamine (15 mg kg21). Rabbits were placed on a heated operating table (Harvard Apparatus, Boston, MA, USA) to maintain normothermia. Temperature was monitored by a rectal probe and kept in a normal range (38 – 398C) by heating lamps. A catheter was placed into the left or right ear vein for maintenance of anaesthesia with fentanyl (7.5 mg kg21). Subsequently, animals were ventilated with oxygen 100% by a face mask to avoid hypoxia and underwent tracheostomy. A tracheal tube (Ø 3.0 mm, Vygon GmbH, Aachen, Germany) was inserted for continuous artificial ventilation (Inspira advanced safety ventilator, Harvard Apparatus) using oxygen 100% during further preparation. A central venous catheter was inserted into the jugular vein. Anaesthesia was maintained i.v. using thiopental (30 mg kg21 h21) and fentanyl (40 mg kg21 h21) throughout the whole experiment. For MAP measurements and blood gas analysis, a catheter was placed into the left femoral artery. Adequate anaesthesia was ascertained by measurement of MAP and detection of possible MAP changes throughout the experiment. A further arterial line was inserted into the descendent aorta via the right femoral artery for blood withdrawal for measurement of microspheres. A left-sided thoracotomy was performed through the third to fifth rib 0.5 cm lateral to the sternum and the pericardium was opened. For later injections of coloured microspheres, a fluid-filled catheter was inserted into the left atrium through the atrial appendage and secured with a 6-0 silk suture. Subsequently, a 4-0 silk suture was placed around the large marginal branch of the circumflex coronary artery near the left atrial appendage. By threading the two ends of the suture through a small plastic tube, a loose snare was formed allowing CAO. At the end of the surgical preparation, a stabilization period was allowed for 20 min and inspired oxygen concentration was reduced to 30%.
(RPP) by the equation:10
Haemoglobin solution reduces infarct size
buffered 2,3,5-triphenyltetrazolium chloride (TTC) (Sigma Chemicals) and then immersed in 10% buffered formaldehyde.12 – 14 Again, a scan was performed to distinguish the normal (TTC-positive) myocardium from the infarcted (TTC-negative) fraction of the myocardium. The area at risk was expressed as the percentage of the left ventricle. The area of impaired perfusion and the TTC-negative area were expressed as the percentage of the area at risk. The variables were calculated by computerized planimetry (ScionImageTM , Scion Corp., Frederick, MD, USA) by an investigator who was blinded with regard to the respective group.
Regional myocardial blood flow
RMBF ¼
Fr AUm AUr
where RMBF is regional myocardial blood flow (ml min21); Fr, the withdrawal rate of the reference arterial sample
Statistics The study was designed to test whether or not the prophylactic or therapeutic application of HBOC-200 reduces the myocardial infarct size after 240 min of reperfusion in comparison with the placebo-treated control group (G3). In addition to the primary endpoint, the effects on RMBF and areas of impaired perfusion were assessed and compared with the control group (G3). On the basis of the hypothesis for the primary study endpoint, a sample size calculation was performed (Instat, Graphpad, CA, USA). We set a 45% infarct size in the ‘area at risk’ as a predicted value for the control group. The smallest difference we decided to be clinically significant was defined as 30%. We permitted a type I error of a¼0.05, and with the alternate hypothesis, the null hypothesis would be retained with a type II error of b¼0.2. Sample size analysis resulted in the need of eight animals per group undergoing the whole study protocol. In case of animal dying before completion of the protocol, the randomization number was used again. Statistical analysis was performed using SPSS 9.0 (SPSS Inc.) and Instat 2.1 (Graphpad Inc.). Statistics for infarct size, area at risk, and impaired perfusion were performed using analysis of variance (ANOVA) with Dunnet’s post hoc test (vs G3). Continued variables were analysed using ANOVA with Bonferroni’s post hoc test. Data are presented as mean (SD) unless otherwise indicated. A P-value of ,0.05 was considered to be statistically significant.
Results Mortality and exclusions One animal in the prophylactic group died during coronary occlusion because of a severe arrhythmia. Thirty-two animals completed the whole study (eight in each group).
Haemodynamics Baseline values of heart rate and MAP were comparable between all groups (Fig. 2). Treatment with HBOC-200 in a prophylactic (G1) and therapeutic manner (G2) decreased heart rate and increased MAP significantly. For heart rate, the effect was almost apparently constant in comparison with the positive control group (G3) beginning at the end of ischaemia until the end of experiment. A significant difference in heart rate was also intermittently noticed after administration of HBOC-200 (G1) in comparison with the negative control group (G4). Elevated
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Coloured plastic microspheres (yellow and violet; Dye-trakw, Triton Technology, San Diego, CA, USA) were used to measure RMBF as previously described.15 Microspheres were 15 (1) mm in size and suspended in 10% Tween 80. To ensure adequate dispersion, solution of microspheres with a concentration of 750 000 spheres per millilitre was continuously rotated in a rotator during the whole experiment and vortexed immediately before use (VF2, Jane und Kunkel, Stauffer, Germany) and subsequently ultrasonically homogenized for 1 min (Son plus HD 2070, Baseline electronics, Berlin, Germany). RMBF was measured by intra-atrial injection of 750 000 microspheres per measurement at 20 min after CAO and after 2 min of reperfusion. Simultaneously, a reference blood sample was withdrawn with a special suction device (11 plus, Harvard Medical Instruments, Holliston, MA, USA) through the right arterial catheter at a rate of 2.0 ml min21. When the withdrawn blood appeared in the arterial line, microsphere injection was started immediately over a period of 30 s. Blood withdrawal was continued for 2 min after the injection. Each microsphere injection was subsequently followed by a flush of 2 ml saline solution over a period of 30 s. At the end of the experiment, tissue samples from the ischaemic area of the apex of the heart were obtained according to the in vivo staining of the blue dye. Microspheres from tissue and blood were recovered by digestion in a 1.0 M KOH solution at 508C overnight and subsequently processed and evaporated the next day according to the instructions provided by the manufacturer.16 At the end of the process, dyes were recovered from the spheres within 200 ml dimethylformamide, and their concentrations were determined by spectrophotometry at fixed wavelengths for each dye according to the manufacturer’s instructions. The composite spectrum of each dye solution was resolved into the spectra of the single constituents by a matrix inversion technique.17 RMBF was calculated using the equation:
(ml min21); AUm, absorbance in the myocardial sample; and AUr, absorbance in the reference blood sample. The amount of myocardial flow per gram of tissue was calculated by dividing the flow by the wet weight of the appropriate tissue sample.
Rempf et al.
140
300 #* #*
130
*
§*
#*
#*
#*
#*
# #§*
Negative control *
Positive control Therapy
250
*
120
Prophylaxis
110
200
100 90
$§*
150 $
*
$* *
80
#* *
*
*
*
*
*
*
100
*
70 60
Heart rate (beats min–1)
Mean arterial pressure (mm Hg)
#
# #§*
50 40
0 00:10
00:25
00:45
01:00
01:12
01:30
01:50
02:10
03:10
04:10
05:10
Time: hh:mm
Fig 2 Plot of haemodynamic data over time. Black arrows indicate time of HBOC-200 application. P,0.05: *positive control (G3) vs prophylaxis (G1), #positive control (G3) vs therapy (G2), §negative control (G4) vs prophylaxis (G1), $therapy (G2) vs prophylaxis (G1).
Table 1 RPP data over time [MW (SD), *P,0.05 vs prophylaxis and positive control]. Black arrows indicate the time of HBOC-200 application RRP (mm Hg)
! Coronary artery occlusion
!
Reperfusion
Time (hh:mm)
Negative control
Positive control
Therapy
Prophylaxis
00 : 10 00 : 15 00 : 25 00 : 35
23.1 (2) 24.6 (3) 24.5 (2) 25.2 (2)
24.5 23.4 24.7 25.8
(3) (3) (3) (3)
23.0 23.1 23.5 23.7
(3) (3) (3) (3)
25.5 (6) 23.9 (6) 23.3 (7) 22.5 (7)
00 : 45 00 : 50 01 : 00 01 : 05
24.1 (2) 24.9 (2) 24.8 (3) 25.3 (3)
24.5 21.8 25.3 25.8
(5) (3) (4) (3)
23.8 19.2 22.3 20.4
(4) (5) (5) (4)
22.9 (7) 22.5 (6) 21.6 (5) 22.9 (6)
01 : 12 01 : 20 01 : 30 01 : 40 01 : 50 02 : 00 02 : 10 02 : 40 03 : 10 03 : 40 04 : 10 04 : 40 05 : 10
25.4 (4) 27.3 (3)* 25.7 (2) 24.9 (4) 24.9 (1) 24.4 (1) 25.1 (2) 24.8 (4) 25.6 (3) 25.4 (3) 25.1 (2) 25.5 (3) 23.9 (5)
23.7 23.6 22.9 23.7 24.4 24.3 24.9 25.5 24.0 24.5 24.2 24.9 23.8
(4) (3) (4) (3) (3) (3) (3) (3) (3) (3) (3) (3) (3)
19.8 22.1 20.3 21.6 21.6 22.1 21.5 21.4 21.4 21.7 20.1 22.4 22.5
(3) (4) (3) (4) (4) (4) (3) (3) (3) (2) (5) (3) (5)
21.1 (5) 21.4 (4) 22.1 (6) 22.2 (5) 22.6 (4) 22.6 (4) 23.1 (5) 24.2 (4) 24.2 (4) 22.6 (2) 21.7 (4) 24.1 (3) 23.6 (5)
MAP was observed immediately after prophylactic administration of HBOC-200 (G1) in comparison with all other groups (G2 –4). However, in contrast to results of the heart rate, this episode was only significant until 90 min before the end of experiment compared with the positive control group (G3). When HBOC-200 was administered in the therapy group (G2), a similar trend for MAP was noted; nevertheless, the difference did reach significance only 10 min after reperfusion in comparison with the positive control (G3). Although HBOC-treated groups (G1 and 2)
exhibited lower heart rates and elevated MAPs, the RPP of all ischaemic groups (G1– 3) were comparable over the time of experiment with the exception of RPP being higher in the negative control group compared with G1 and 2 once 15 min after reperfusion (Table 1).
Blood gas analysis and temperature There were no significant differences between the groups with respect to PCO2, PO2, pH, BE, Naþ, Kþ, and
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50
Haemoglobin solution reduces infarct size
Area at risk, infarct size, and areas of impaired perfusion (NR) The average risk area in the three ischaemic groups (G1–3) ranged between 20% and 25% of the left ventricle [G1, 21 (9)%; G2, 23 (8)%; G3, 25 (15)%]. The negative control group (G4) experienced slightly smaller risk areas [16 (5)%]. However, there were no significant differences between the groups with respect to the area at risk. After 30 min of coronary occlusion and 240 min of reperfusion, the area at risk that developed infarction was 48 (17)% in the positive control group (G3), whereas no infarction was detected by TTC staining in the negative control (G4) except minor zones adjacent to the suture. Administration of HBOC-200 in a prophylactic and therapeutic manner (G1 and 2) reduced infarct size significantly to about 25% of the area at risk [G1, 25 (13)%; G2, 22 (20)%, P,0.05]. The anatomic zone of areas of impaired perfusion was delineated by thioflavin-S staining. In positive control rabbits, 35 (12)% of the area at risk showed a perfusion defect (NR phenomenon). Minor perfusion defects in the negative control group (G4) corresponded again to the suture and averaged 5 (3)% of the area at risk (P,0.008 vs G3). The extent of areas of impaired perfusion as percentage of the area at risk was not significantly
Table 2 Blood gases and haemoglobin concentrations during experimental protocol. Data are mean (SD). PaCO2 and PaO2, arterial partial pressure of carbon dioxide and oxygen, respectively; Hbt, total haemoglobin concentration; MetHb, met-haemoglobin; HbO2, oxyhaemoglobin; Hbp, plasma haemoglobin. Appl., application; Occl., occlusion; Rep., reperfusion. P,0.05: *positive control vs prophylaxis, #positive control vs therapy, §negative control vs prophylaxis, $therapy vs prophylaxis, †negative control vs therapy Time (hh:mm)
Coronary artery occlusion 00 : 30 Baseline
PaCO2 (mm Hg) Prophylaxis Therapy Positive control Negative control PaO2 (mm Hg) Prophylaxis Therapy Positive control Negative control Hbt (g dl21) Prophylaxis Therapy Positive control Negative control MetHb (g dl21) Prophylaxis Therapy Positive control Negative control HbO2 (%) Prophylaxis Therapy Positive control Negative control HbP (g dl21) Prophylaxis Therapy Positive control Negative control
35 35 35 34
(6) (3) (5) (5)
00 : 45 Appl. 1110 min
41 36 35 34
(5) (3) (5) (5)
01 : 05 Occl. 15 min
37 35 35 34
(4) (3) (5) (5)
01 : 20 Appl. 2110 min
36 38 36 34
(2) (5) (6) (2)
01 : 32 Rep. 12 min
35 34 35 34
(2) (13) (5) (3)
05 : 30 Rep. 1240 min
38 39 38 41
(6) (4) (3) (13)
124 (28) 115 (25) 120 (19) 109 (33)
113 (27) 106 (23) 118 (24) 126 (33)
126 106 120 116
(25) (19) (22) (24)
166 (98) 141 (63) 180 (127) 111 (22)
147 (50) 104 (20) 109 (20) 113 (24)
142 (29) 166 (110) 138 (31) 162 (99)
13.2 (1.0) 13.0 (0.8) 12.9 (0.6) 12.2 (0.6)
12.5 (0.9) 12.1 (1.1) 11.9 (1.0) 11.7 (0.5)
12.5 12.3 12.0 12.0
(1.1) (0.8) (1.0) (0.5)
12.2 (0.9) 12.0 (0.4) 11.6 (0.9) 11.7 (0.5)
11.9 (0.9) 11.8 (0.7) 11.3 (1.0) 10.8 (1.0)
11.2 (0.9) 11.4 (1.2) 11.4 (0.7) 10.3 (1.1)
0.7 (0.1) 0.6 (0.1) 0.6 (0.2) 0.7 (0.1)
0.8 (0.3) 0.6 (0.1) 0.6 (0.2) 0.6 (0.1)
0.8 0.7 0.6 0.6
(0.2) (0.1) (0.2) (0.1)
0.8 (0.1) 0.7 (0.2) 0.6 (0.0) 0.6 (0.1)
0.7 (0.2) 0.7 (0.1) 0.8 (0.1) 0.6 (0.1)
96.2 (1.1) 95.4 (1.8) 96.0 (1.1) 95.5 (2.1)
94.4 (1.0) 95.0 (1.5) 95.7 (1.4) 97.1 (1.1)§
94.9 95.0 95.9 96.4
(1.3) (1.6) (1.2) (0.8)
95.4 (0.9)$ 92.6 (2.6) 95.1 (1.5) 96.1 (1.5)†
95.6 (1.6) 93.3 (2.9) 95.7 (1.3) 96.6 (1.1)†
1.2 (0.1) 0 (0)$ 0 (0)* 0 (0)§
1.2 0 0 0
(0.1) (0)$ (0)* (0)§
1.0 (0.1) 1.1 (0.1) 0 (0)*,# 0 (0)§,†
0.8 (0.1) 0.8 (0.1) 0 (0)*,# 0 (0)§,†
0 0 0 0
(0) (0) (0) (0)
501
0.8 (0.2) 0.8 (0.2)# 0.6 (0.1) 0.7 (0.1) 95.6 (0.9) 93.0 (6.1) 96.0 (1.2) 95.8 (1.5) 1.1 (0.1) 1.1 (0.1) 0 (0)*,# 0 (0)§,†
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glucose concentration (Table 2). The total haemoglobin and carboxyhaemoglobin concentrations did not differ between the groups during the whole observation period. HBOC-200 treated groups (G1 and 2) had methaemoglobin levels up to a peak concentration of 0.8 g dl21. However, the difference only reached significance in the therapeutic group (G2) in comparison with the positive control group (G3) after 20 min of coronary occlusion. The oxyhaemoglobin concentration in G4 was significantly higher in comparison with the prophylactic group (G1) after HBOC-200 application. During reperfusion, the oxyhaemoglobin concentration in the therapeutic group (G2) was significantly reduced in comparison with the negative control group (G4). At the beginning of reperfusion, the oxyhaemoglobin concentration in the prophylactic group (G1) was significantly higher in comparison with the therapy group (G2). The administration of HBOC-200 was accompanied by the expected elevated plasmatic haemoglobin concentration up to a maximum of 1.3 g dl21, followed by a moderate decrease in the prophylactic and the therapeutic groups (G1 and 2). The body core temperature was similar in all groups.
Rempf et al.
affected by therapeutic [G2, 34 (22)%] or prophylactic [G1, 26 (15)%] HBOC-200 treatment in comparison with the positive control group (G3).
Table 3 RMBF in the area at risk (blue dye negative areas) of the apex of the heart (ml min21 g21) at 25 min of coronary occlusion and 2 min of reperfusion Prophylaxis
Therapy
Positive control
Negative control
0.05 (0.04)
0.11 (0.07)
0.05 (0.02)
1.91 (0.7)
3.21 (1.36)
2.84 (0.56)
2.92 (1.41)
2.11 (0.54)
Regional myocardial blood flow During occlusion, all groups were equally ischaemic in the risk area with blood flow close to zero (Table 3). In all ischaemic groups, average blood flow in the previously ischaemic region increased after 2 min of reperfusion. No statistically significant difference between the groups could be observed.
Discussion
after reperfusion was significantly improved with HBOC-201 treatment in comparison with the saline control group. In the second model, called the myocardial infarction model, ischaemia was similarly created by LAD stenosis to 80– 90% flow reduction with concurrent cardiac pacing at a rate of 10% above spontaneous heart rate. Ischaemia was maintained for 195 min and reperfusion was allowed for 180 min. Dogs were treated during ischaemia at different times with different doses of HBOC-201 (0.5 and 1 g kg21 at 15 min ischaemia and 1 g kg21 at 60 min ischaemia). Again, no evidence of impaired coronary flow or significant coronary vasoconstriction was found in animals treated with HBOC-201. When 1 g kg21 HBOC-201 was given 15 min after the onset of ischaemia HBOC, an increase in LAD flow in comparison with the control groups was reported. The mean coronary resistance was lowered in HBOC-201-treated animals after 60 min of ischaemia in comparison with the saline control group, and myocardial infarct size as percentage of the area at risk was significantly reduced when 1 g kg21 HBOC-201 was given 15 min after ischaemia in comparison with the control groups. Furthermore, George and colleagues20 reported that infarct size in the heart layer most vulnerable to ischaemia was significantly diminished in animals with delayed, modest, and enlarged HBOC-201 treatment. One of our previous studies13 showed that prophylactic application of 0.4 g kg21 before acute LAD occlusion (25 min) and reperfusion (120 min) reduced infarct size, density of DNA single-strand breaks, and severity of cardiac arrhythmias compared with a saline-treated control group in rats. However, when given during LAD occlusion before reperfusion, HBOC-200 failed to show beneficial effects.13 Results of the present study show that both prophylactic and therapeutic treatment with HBOC-200 is effective to reduce myocardial infarct size, in comparison with the saline-treated positive control group after 30 min of CAO and 240 min of reperfusion in an animal model of low collateralization. Although reasons for these different results are unknown, the duration of reperfusion may have an impact (120 vs 240 min of reperfusion). In the present study, ischaemia and reperfusion were confirmed by a microsphere technique. All treated groups were equally ischaemic in comparison with the positive control and no animal showed a failure of the coronary artery reperfusion at 2 min of
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In a recent meta-analysis including all available clinical trials evaluating artificial oxygen carriers like HBOC-200, increased risk of mortality and myocardial infarction was found,7 18 and this led to a demand that any new or existing HBOC should be subjected to preclinical studies in animal models that replicate the known toxicities of HBOC demonstrated in humans before further clinical trials of this class of product are allowed to proceed. In contrast to the findings of the recent meta-analysis, experimental animal studies investigating the effects of HBOC-200 and HBOC-201 on infarct size were able to show that both HBOC solutions can reduce myocardial infarct size in animal models.13 19 20 Caswell and colleagues,19 in their study of HBOC-201 pre-ischaemic-treated dogs (10% of total blood volume was removed and replaced with HBOC-201), found a reduction in creatine kinase MB isoform release, a reduction in neutrophil infiltration in the area at risk, and a 56% reduction in myocardial infarct size in comparison with a saline-treated control group after 90 min of ischaemia and 270 min of reperfusion. Ischaemia was performed by complete left anterior descending (LAD) occlusion. RMBF from the epicardium and endocardium during baseline was comparable between HBOC and saline-treated dogs. At ischaemia, both groups showed a significant RMBF decrease in comparison with baseline values without significant differences between saline- and HBOC-201-treated animals. George and colleagues20 investigated in two distinct canine models the effects of HBOC-201 on LAD flow, regional myocardial function, RMBF, and myocardial infarct size. Ischaemia was produced by acute LAD stenosis to 80– 95% flow reduction from baseline. In the first model, called the regional ischaemia model, the stenosis was maintained for a total of 60 min followed by 15 min of reperfusion. After 15 min of ischaemia, dogs were treated with 1 g kg21 HBOC-201. Compared with a saline and phenylephrine control group, LAD flow improved and mean LAD resistance was lowered significantly in the HBOC-201-treated group. Additionally, RMBF in the area at risk was significantly higher with HBOC-201 treatment at the end of ischaemia and regional myocardial function
25 min coronary artery occlusion 2 min reperfusion
Haemoglobin solution reduces infarct size
after 240 min of reperfusion, suggesting that HBOC-200 has no beneficial effect on NR areas. It has been suggested that bovine haemoglobin given before ischaemia may reduce the severity of ischaemia by preloading the myocardium with oxygen or by aiding in oxygen delivery to hypoxic tissue.13 19 20 However, our recent data suggest that therapeutic treatment was as effective as the prophylactic approach to reduce myocardial infarct size in an animal model with poor collateralization. Thus, it appears that the mechanism of action also depends on the time of reperfusion. A potential mechanism for the reduction in infarct size after myocardial ischaemia could be improved oxygenation in the respective tissue. The enhanced oxygen off-loading capacity represents an important part of oxygen delivery responsible for tissue oxygenation by HBOC. HBOC solutions have a high oxygen diffusion coefficient in comparison with whole blood3 and also provide increased oxygen off-load from the red blood cells.4 The augmented oxygen extraction in the presence of HBOC provides enhanced oxygen supply at the tissue site. When compared with red blood cells,30 HBOC seems to have a higher oxygenation potency due to the plasmatic oxygen transport and enhanced oxygen release from HBOC and from adjacent erythrocytes. In this context, the damaged myocardial tissue seems to benefit from the oxygenation with HBOC-200 in the present model of myocardial ischaemia and reperfusion, which may result in a decreased infarct size by prophylactic and therapeutic treatment.
Conclusion We were able to show that prophylactic and therapeutic application of HBOC-200 reduces infarct size in a myocardial ischaemia and reperfusion model in rabbits. This reduction of infarct size is not accompanied by a reduction of areas of impaired perfusion. Therefore, clinical trials with a low dosage of HBOC to provide improved oxygen delivery in an organ of impaired perfusion could be of interest.
Funding This work was completely financed by the Department of Anaesthesiology, University Hospital Hamburg Eppendorf, Germany.
References
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1 Haney CR, Buehler PW, Gulati A. Purification and chemical modifications of hemoglobin in developing hemoglobin based oxygen carriers. Adv Drug Deliv Rev 2000; 40: 153– 69 2 Soma LR, Uboh CE, Guan F, et al. The pharmacokinetics of hemoglobin-based oxygen carrier hemoglobin glutamer-200 bovine in the horse. Anesth Analg 2005; 100: 1570– 5
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reperfusion. However, in the present study, we did confirm an alteration on haemodynamics by HBOC-200 treatment in our model of myocardial infarction and reperfusion. When HBOC-200 was given before CAO, a significant increase in MAP and a decrease in heart rate were observed. The effect of MAP was less obvious when HBOC-200 was given during ischaemia. Therefore, the observed infarct size reduction by HBOC-200 might be attributable to changes on myocardial oxygen consumption by haemodynamic parameters. However, the RPP for all ischaemic groups was only significantly different at one time after reperfusion. One limitation of our study is that, like in all other animal studies, rabbits with normal coronary arteries and without significant collateral coronary blood flow are not necessarily comparable with humans with significant coronary artery disease dependent on collateral flow to prevent extensive myocardial infarction. Another limitation could be in the limited time of observation of the animals. In many clinical trials, with the use of higher dosages of HBOC administered in the perioperative period, myocardial infarctions in humans were identified over a period of days instead of immediately after reperfusion. Results of a possible administration of a higher dosage of HBOC in our trial are also unknown. Additionally, the study is limited, in that we did not use another control group of non-oxygen carrying solution with similar colloid osmotic pressure, as the changes we detected probably may not directly be related to the oxygen carrying capacity of HBOC. As previously described, factors like a high colloid osmotic pressure resulting in a haemodilution by intravascular fluid shift may have an influence on our results, too.21 There is evidence from animal studies that the vasoconstrictive side-effects associated with HBOC treatment are caused by nitric oxide scavenging, endothelin release, or modulation of alpha-adrenoreceptors.22 – 24 This vasoconstrictive effect of cell-free haemoglobin solutions might be deleterious in circumstances of areas of impaired perfusion (NR) after myocardial ischaemia and reperfusion. It is known that a part of the myocardial capillary network after ischaemia is irreversibly closed at the time of reperfusion, despite restoration of epicardial vessel patency.25 The extent of resulting areas of NR depends on the length of ischaemia and the effects continue during reperfusion.11 This ‘NR phenomenon’ is known to be a consequence of a complex interaction of endothelial cell damage, vascular dysfunction, and perivascular swelling and also microembolism by platelets and leucocytes and their interactions with the endothelium.25 Studies in animals11 25 26 and clinical investigations27 28 demonstrated a close correlation between areas of impaired perfusion and myocardial necrosis. Furthermore, it has been described that NR predicts infarct size expansion.29 In the present study, the areas of NR in the area at risk were not significantly different from HBOC-200 treated and untreated hearts
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regional blood flow using the radioactive microsphere method. Comput Programs Biomed 1979; 9: 19 – 38 Levien LJ, Hodgson RE, James MF. Hemoglobin-based blood substitutes and risk of myocardial infarction and death. J Am Med Assoc 2008; 300: 1295; author reply 1298 – 9 Caswell JE, Strange MB, Rimmer DM, III, Gibson MF, Cole P, Lefer DJ. A novel hemoglobin-based blood substitute protects against myocardial reperfusion injury. Am J Physiol Heart Circ Physiol 2005; 288: H1796– 801 George I, Yi GH, Schulman AR, et al. A polymerized bovine hemoglobin oxygen carrier preserves regional myocardial function and reduces infarct size after acute myocardial ischemia. Am J Physiol Heart Circ Physiol 2006; 291: H1126– 37 Driessen B, Jahr JS, Lurie F, Griffey SM, Gunther RA. Effects of haemoglobin-based oxygen carrier hemoglobin glutamer-200 (bovine) on intestinal perfusion and oxygenation in a canine hypovolaemia model. Br J Anaesth 2001; 86: 683 – 92 Sharma AC, Gulati A. Yohimbine modulates diaspirin crosslinked hemoglobin-induced systemic hemodynamics and regional circulatory effects. Crit Care Med 1995; 23: 874 – 84 Schultz SC, Grady B, Cole F, Hamilton I, Burhop K, Malcolm DS. A role for endothelin and nitric oxide in the pressor response to diaspirin cross-linked hemoglobin. J Lab Clin Med 1993; 122: 301– 8 Rooney MW, Hirsch LJ, Mathru M. Hemodilution with oxyhemoglobin. Mechanism of oxygen delivery and its superaugmentation with a nitric oxide donor (sodium nitroprusside). Anesthesiology 1993; 79: 60 – 72 Reffelmann T, Kloner RA. The ‘no-reflow’ phenomenon: basic science and clinical correlates. Heart 2002; 87: 162 – 8 Ambrosio G, Weisman HF, Mannisi JA, Becker LC. Progressive impairment of regional myocardial perfusion after initial restoration of postischemic blood flow. Circulation 1989; 80: 1846– 61 Ragosta M, Camarano G, Kaul S, Powers ER, Sarembock IJ, Gimple LW. Microvascular integrity indicates myocellular viability in patients with recent myocardial infarction. New insights using myocardial contrast echocardiography. Circulation 1994; 89: 2562– 9 Ito H, Iwakura K, Oh H, et al. Temporal changes in myocardial perfusion patterns in patients with reperfused anterior wall myocardial infarction. Their relation to myocardial viability. Circulation 1995; 91: 656 – 62 Reffelmann T, Hale SL, Dow JS, Kloner RA. No-reflow phenomenon persists long-term after ischemia/reperfusion in the rat and predicts infarct expansion. Circulation 2003; 108: 2911 –7 Standl T, Horn P, Wilhelm S, et al. Bovine haemoglobin is more potent than autologous red blood cells in restoring muscular tissue oxygenation after profound isovolaemic haemodilution in dogs. Can J Anaesth 1996; 43: 714 – 23
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3 Budhiraja V, Hellums JD. Effect of hemoglobin polymerization on oxygen transport in hemoglobin solutions. Microvasc Res 2002; 64: 220 – 33 4 Page TC, Light WR, Hellums JD. Prediction of microcirculatory oxygen transport by erythrocyte/hemoglobin solution mixtures. Microvasc Res 1998; 56: 113 – 26 5 Kasper SM, Grune F, Walter M, Amr N, Erasmi H, Buzello W. The effects of increased doses of bovine hemoglobin on hemodynamics and oxygen transport in patients undergoing preoperative hemodilution for elective abdominal aortic surgery. Anesth Analg 1998; 87: 284 – 91 6 Winslow RM. Alphaalpha-crosslinked hemoglobin: was failure predicted by preclinical testing? Vox Sang 2000; 79: 1 – 20 7 Natanson C, Kern SJ, Lurie P, Banks SM, Wolfe SM. Cell-free hemoglobin-based blood substitutes and risk of myocardial infarction and death: a meta-analysis. J Am Med Assoc 2008; 299: 2304– 12 8 National Research Council IoLAR, Commission on Life Sciences. Guide for the Care and Use of Laboratory Animals. Washington, DC: National Academy Press, 1996 9 Moallempour M, Jahr JS, Lim JC, Weeks D, Butch A, Driessen B. Methemoglobin effects on coagulation: a dose –response study with HBOC-200 (Oxyglobin) in a thrombelastogram model. J Cardiothorac Vasc Anesth 2009; 23: 41– 7 10 Wilkinson PL, Moyers JR, Ports T, Chatterjee K, Ullyott D, Hamilton WK. Rate-pressure product and myocardial oxygen consumption during surgery for coronary artery bypass. Circulation 1979; 60: 170 –3 11 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 12 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 13 Burmeister MA, Rempf C, Standl TG, et al. Effects of prophylactic or therapeutic application of bovine haemoglobin HBOC-200 on ischaemia – reperfusion injury following acute coronary ligature in rats. Br J Anaesth 2005; 95: 737– 45 14 Rempf C, Standl T, Gottschalk A, et al. Effects of a prophylactic or therapeutic application of perflubron emulsion on myocardial ischaemia–reperfusion injury in rats. Eur J Anaesthesiol 2008; 25: 850–9 15 Hale SL, Vivaldi MT, Kloner RA. Fluorescent microspheres: a new tool for visualization of ischemic myocardium in rats. Am J Physiol 1986; 251: H863– 8 16 Kowallik P, Schulz R, Guth BD, et al. Measurement of regional myocardial blood flow with multiple colored microspheres. Circulation 1991; 83: 974 –82 17 Schosser R, Arfors KE, Messmer K. MIC-II—a program for the determination of cardiac output, arterio-venous shunt and
doi:10.1093/bja/aep233
Advance Access publication August 26, 2009
Administration of bovine polymerized haemoglobin before and during coronary occlusion reduces infarct size in rabbits‡ C. Rempf1, T. Standl2, K. Schenke3, K. Chammas4, A. Gottschalk5, M.-A. Burmeister6† and A. Gottschalk7* 1
*Corresponding author. E-mail: [email protected] 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 kg21 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)%, P¼0.026] and G2 [22 (20)%, P¼0.009] 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. Br J Anaesth 2009; 103: 496–504 Keywords: heart, coronary occlusion; heart, ischaemia; model, rabbit Accepted for publication: July 7, 2009
Cell-free haemoglobin-based oxygen carriers (HBOC) were primarily developed for replacement of red blood cells. The haemoglobin-glutamer 200 (HBOC-200; Oxyglobinw) is a glutaraldehyde polymerized bovine haemoglobin. Glutaraldehyde works as a cross-linking agent, which stabilizes the tetramer form of haemoglobin by non-specific modifications to both a- and b-globin chains and then as a protein surface polymerizing agent that produces a polymer.1 2
In contrast to the veterinary product Oxyglobinw (HBOC-200), the solution Hemopurew (HBOC-201) is approved for sale in South Africa for the treatment of †
Declaration of interest. M.-A.B. is employed by B. Braun Melsungen AG, Melsungen, Germany, an international Medical Device and Pharmaceutical Manufacturer. ‡ This article is accompanied by Editorial III.
# The Author [2009]. Published by Oxford University Press on behalf of the British Journal of Anaesthesia. All rights reserved. For Permissions, please email: [email protected]
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Department of Anaesthesiology, University Medical Center Schleswig-Holstein, Campus Lu¨beck, Germany. 2 Department of Anaesthesiology and Critical Care Medicine, Academic Hospital Solingen, Germany. 3 Department of Cardiology and Pneumology, Altona General Hospital, Hamburg, Germany. 4Department of Radiology, Children’s Hospital Altona, Hamburg, Germany. 5Department of Anaesthesiology and Intensive Care Medicine, University Hospital Muenster, Germany. 6Department of Anaesthesiology, University Hospital Hamburg Eppendorf, Germany. 7Department of Anaesthesiology, Intensive Care Medicine and Pain Therapy, Knappschaftskrankenhaus Bochum Langendreer, University Hospital Bochum, Germany
Haemoglobin solution reduces infarct size
Methods This study was approved by the local committee for animal care (Behoerde fu¨r Arbeit, Gesundheit und Soziales,
Hamburg, Germany) in accordance with the Guide for the Care and Use of Laboratory Animals.8
Experimental protocol The study protocol (Fig. 1) was designed to assess the influence of prophylactic and therapeutic administration of HBOC-200 on infarct size and areas of impaired perfusion after 30 min of coronary artery occlusion (CAO) and 240 min of reperfusion in an animal model of low coronary collateralization. The rabbits were randomized to one of the four groups. Animals received either 0.4 g kg21 HBOC-200 25 min before ischaemia (G1 prophylaxis, n¼8) or 0.4 g kg21 HBOC-200 10 min after ischaemia (G2 therapy, n¼8). Control groups obtained the equivalent volume of saline with ischaemia and reperfusion for positive controls (G3, n¼8) and without ischaemia and reperfusion for negative controls (G4, n¼8). All fluids were infused via the ear vein over a 5 min period.
Study solution HBOC-200 (Oxyglobinw, Biopure, Cambridge, MA, USA) is based on glutaraldehyde-polymerized, ultrapurified bovine haemoglobin formulated in a modified lactated Ringer’s solution. It contains 13.0 g dl21 of cell-free haemoglobin with a p50 (oxygen affinity) of 36 mm Hg. The oncotic pressure of HBOC-200 is similar to 5% albumin, and the osmolality is similar to normal plasma. HBOC-200 used in our study was bought from a veterinary pharmacy. HBOC bags were stored in a special box as HBOC rapidly oxidizes to met-haemoglobin with consequences
Group 1: prophylaxis: n=8 HBOC Baseline
NaCl Ischaemia 30 min
Reperfusion 240 min
HBOC Ischaemia 30 min
Reperfusion 240 min
NaCl Ischaemia 30 min
Reperfusion 240 min
Group 2: therapy: n=8 NaCl Baseline Group 3: positive control: n=8 NaCl Baseline Group 4: negative control: n=8 NaCl
NaCl
Baseline time (h:mm) 0:00 0:05 0:10 0:15 0:20 0:25 0:30 0:35 0:40 0:45 0:50 0:55 1:00 1:05 1:10 1:12
BGA
BGA
BGA
BGA RMBF
BGA RMBF
Fig 1 Schematic overview of study design. BGA, blood gas analysis; RMBF, regional myocardial blood flow.
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5:20
BGA Thioflavin Patent blue Infarct size
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acutely anaemic surgical patients. Both solutions are similar with the exception of absolute molecular weight distribution of the polymers. It seems that haemoglobin in HBOC may be transported in blood plasma to areas where red blood cell flow is not adequate. Further, enhanced oxygen off-loading through capillaries might be beneficial to patients with cardiovascular risks.3 4 However, these artificial oxygen carriers increase mean arterial pressure (MAP) and pulmonary artery pressure and also systemic and pulmonary vascular resistance.5 6 This property of cell-free haemoglobin solutions might be harmful in patients with coronary heart disease. A recent meta-analysis including all available clinical HBOC trials found an increased risk of mortality and myocardial infarction.7 HBOC-induced vasoconstriction may increase areas of impaired perfusion, so-called ‘no reflow’ (NR) phenomenon, which may compromise tissue oxygen supply. The NR phenomenon is observed after transient coronary occlusion and characterized by areas of hypoperfusion and decreased return of regional myocardial blood flow (RMBF). Since only very limited data exist, we performed a prospective, randomized, and blinded animal trial to evaluate the effects of HBOC-200 given in a prophylactic and therapeutic manner on myocardial infarct size and ischaemia/reperfusion-related areas of impaired perfusion. We hypothesized that the administration of HBOC results in a decrease in infarct size and areas of impaired perfusion.
Rempf et al.
on oxygen binding and coagulation parameters.9 After closure of the box, air was evacuated and simultaneously the box was filled with nitrogen for 5 min. Safe closure of the box was confirmed using a negative pressure within the box. Before using the HBOC in the next animal, met-haemoglobin was measured. If met-haemoglobin was .5%, the solution was not accepted and a new bag was opened.
Surgical preparation
Haemodynamics During the whole experiment, heart rate, MAP and systolic arterial pressure, temperature, and pulse oximetry were continuously measured and recorded (Marquette Tramscope 12, Milwaukee, WI, USA). To assess the influence of haemodynamic parameters on myocardial oxygen consumption, we calculated the rate pressure product
RPP ¼
ðheart rateÞ ðarterial systolic blood pressureÞ 1000
Blood gas analysis Arterial blood samples were obtained repetitively in 150 ml glass tubes (Clinitubes, Radiometer Medical, Kopenhagen, Denmark). Measurements included blood gases, electrolytes, and glucose (ABL 505, Radiometer Medical, Copenhagen, Denmark), and concentrations of total haemoglobin (Hb), oxyhaemoglobin (HbO2), carboxyhaemoglobin (COHb), met-haemoglobin (MetHb), and plasmatic haemoglobin (Hbp) (OSM 3, Radiometer Medical, Copenhagen, Denmark). For measurement of plasmatic haemoglobin, 80 ml of arterial blood was centrifuged for 5 min at 2240 g and the plasma was obtained for analysis. Blood samples were obtained at baseline (5 min before prophylactic treatment), 10 min after prophylactic treatment, 5 and 20 min during ischaemia, and 2 and 240 min after reperfusion (Fig. 1).
Measurement of area at risk, impaired perfusion, and infarction At the end of the protocol, the area of impaired perfusion was delineated by an injection of the fluorescent dye thioflavin-S (1 ml kg21, Sigma Chemicals, Munich, Germany) via the left atrial catheter. The dye was prepared as a 4% solution in a warm saline solution, centrifuged for 10 min at 230 g and subsequently filtered to remove particles (pore size 0.2 mm) and injected manually over 20 s. Immediately after injection of thioflavin-S, the coronary artery was re-occluded followed by an injection of a blue dye (Patentblau 1 ml kg21, Sigma Chemicals) through the left atrial catheter to define the area at risk. After 1 min, when the perfused areas of the heart and the lungs were stained blue, the deeply anaesthetized animals were killed by i.v. injection of potassium chloride 7.45% (2 mmol kg21). The heart was rapidly excised and rinsed in cold isotonic saline, placed in a precooled (2208C) heart matrix for rabbits (ASI Instruments, Warren, MI, USA) and frozen for 20 min at 2208C before slicing into 2 mm transverse rings from the apex to the base. The apex of the heart was returned for measurements of RMBF in the area at risk. The remaining slices were first scanned to identify the ‘area at risk’ (uncoloured by the blue dye) and then photographed in the dark under ultraviolet light using a Fluor-STM Multiimager (Bio-Rad, Hercules, CA, USA) to determine areas, which were not perfused by thioflavin-S. Thioflavin-S is a fluorescent vital dye staining the endothelium of the blood vessels,11 and thus the normally perfused myocardium (fluorescent) was easily differentiated from the non-perfused (non-fluorescent) myocardium. Subsequently, slices were incubated for 10 min at 378C in a 1% solution of
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Male New Zealand white rabbits (weight, 3.0 kg) were anaesthetized by i.m. injection of midazolam (1.5 mg kg21) and S-ketamine (15 mg kg21). Rabbits were placed on a heated operating table (Harvard Apparatus, Boston, MA, USA) to maintain normothermia. Temperature was monitored by a rectal probe and kept in a normal range (38 – 398C) by heating lamps. A catheter was placed into the left or right ear vein for maintenance of anaesthesia with fentanyl (7.5 mg kg21). Subsequently, animals were ventilated with oxygen 100% by a face mask to avoid hypoxia and underwent tracheostomy. A tracheal tube (Ø 3.0 mm, Vygon GmbH, Aachen, Germany) was inserted for continuous artificial ventilation (Inspira advanced safety ventilator, Harvard Apparatus) using oxygen 100% during further preparation. A central venous catheter was inserted into the jugular vein. Anaesthesia was maintained i.v. using thiopental (30 mg kg21 h21) and fentanyl (40 mg kg21 h21) throughout the whole experiment. For MAP measurements and blood gas analysis, a catheter was placed into the left femoral artery. Adequate anaesthesia was ascertained by measurement of MAP and detection of possible MAP changes throughout the experiment. A further arterial line was inserted into the descendent aorta via the right femoral artery for blood withdrawal for measurement of microspheres. A left-sided thoracotomy was performed through the third to fifth rib 0.5 cm lateral to the sternum and the pericardium was opened. For later injections of coloured microspheres, a fluid-filled catheter was inserted into the left atrium through the atrial appendage and secured with a 6-0 silk suture. Subsequently, a 4-0 silk suture was placed around the large marginal branch of the circumflex coronary artery near the left atrial appendage. By threading the two ends of the suture through a small plastic tube, a loose snare was formed allowing CAO. At the end of the surgical preparation, a stabilization period was allowed for 20 min and inspired oxygen concentration was reduced to 30%.
(RPP) by the equation:10
Haemoglobin solution reduces infarct size
buffered 2,3,5-triphenyltetrazolium chloride (TTC) (Sigma Chemicals) and then immersed in 10% buffered formaldehyde.12 – 14 Again, a scan was performed to distinguish the normal (TTC-positive) myocardium from the infarcted (TTC-negative) fraction of the myocardium. The area at risk was expressed as the percentage of the left ventricle. The area of impaired perfusion and the TTC-negative area were expressed as the percentage of the area at risk. The variables were calculated by computerized planimetry (ScionImageTM , Scion Corp., Frederick, MD, USA) by an investigator who was blinded with regard to the respective group.
Regional myocardial blood flow
RMBF ¼
Fr AUm AUr
where RMBF is regional myocardial blood flow (ml min21); Fr, the withdrawal rate of the reference arterial sample
Statistics The study was designed to test whether or not the prophylactic or therapeutic application of HBOC-200 reduces the myocardial infarct size after 240 min of reperfusion in comparison with the placebo-treated control group (G3). In addition to the primary endpoint, the effects on RMBF and areas of impaired perfusion were assessed and compared with the control group (G3). On the basis of the hypothesis for the primary study endpoint, a sample size calculation was performed (Instat, Graphpad, CA, USA). We set a 45% infarct size in the ‘area at risk’ as a predicted value for the control group. The smallest difference we decided to be clinically significant was defined as 30%. We permitted a type I error of a¼0.05, and with the alternate hypothesis, the null hypothesis would be retained with a type II error of b¼0.2. Sample size analysis resulted in the need of eight animals per group undergoing the whole study protocol. In case of animal dying before completion of the protocol, the randomization number was used again. Statistical analysis was performed using SPSS 9.0 (SPSS Inc.) and Instat 2.1 (Graphpad Inc.). Statistics for infarct size, area at risk, and impaired perfusion were performed using analysis of variance (ANOVA) with Dunnet’s post hoc test (vs G3). Continued variables were analysed using ANOVA with Bonferroni’s post hoc test. Data are presented as mean (SD) unless otherwise indicated. A P-value of ,0.05 was considered to be statistically significant.
Results Mortality and exclusions One animal in the prophylactic group died during coronary occlusion because of a severe arrhythmia. Thirty-two animals completed the whole study (eight in each group).
Haemodynamics Baseline values of heart rate and MAP were comparable between all groups (Fig. 2). Treatment with HBOC-200 in a prophylactic (G1) and therapeutic manner (G2) decreased heart rate and increased MAP significantly. For heart rate, the effect was almost apparently constant in comparison with the positive control group (G3) beginning at the end of ischaemia until the end of experiment. A significant difference in heart rate was also intermittently noticed after administration of HBOC-200 (G1) in comparison with the negative control group (G4). Elevated
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Coloured plastic microspheres (yellow and violet; Dye-trakw, Triton Technology, San Diego, CA, USA) were used to measure RMBF as previously described.15 Microspheres were 15 (1) mm in size and suspended in 10% Tween 80. To ensure adequate dispersion, solution of microspheres with a concentration of 750 000 spheres per millilitre was continuously rotated in a rotator during the whole experiment and vortexed immediately before use (VF2, Jane und Kunkel, Stauffer, Germany) and subsequently ultrasonically homogenized for 1 min (Son plus HD 2070, Baseline electronics, Berlin, Germany). RMBF was measured by intra-atrial injection of 750 000 microspheres per measurement at 20 min after CAO and after 2 min of reperfusion. Simultaneously, a reference blood sample was withdrawn with a special suction device (11 plus, Harvard Medical Instruments, Holliston, MA, USA) through the right arterial catheter at a rate of 2.0 ml min21. When the withdrawn blood appeared in the arterial line, microsphere injection was started immediately over a period of 30 s. Blood withdrawal was continued for 2 min after the injection. Each microsphere injection was subsequently followed by a flush of 2 ml saline solution over a period of 30 s. At the end of the experiment, tissue samples from the ischaemic area of the apex of the heart were obtained according to the in vivo staining of the blue dye. Microspheres from tissue and blood were recovered by digestion in a 1.0 M KOH solution at 508C overnight and subsequently processed and evaporated the next day according to the instructions provided by the manufacturer.16 At the end of the process, dyes were recovered from the spheres within 200 ml dimethylformamide, and their concentrations were determined by spectrophotometry at fixed wavelengths for each dye according to the manufacturer’s instructions. The composite spectrum of each dye solution was resolved into the spectra of the single constituents by a matrix inversion technique.17 RMBF was calculated using the equation:
(ml min21); AUm, absorbance in the myocardial sample; and AUr, absorbance in the reference blood sample. The amount of myocardial flow per gram of tissue was calculated by dividing the flow by the wet weight of the appropriate tissue sample.
Rempf et al.
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70 60
Heart rate (beats min–1)
Mean arterial pressure (mm Hg)
#
# #§*
50 40
0 00:10
00:25
00:45
01:00
01:12
01:30
01:50
02:10
03:10
04:10
05:10
Time: hh:mm
Fig 2 Plot of haemodynamic data over time. Black arrows indicate time of HBOC-200 application. P,0.05: *positive control (G3) vs prophylaxis (G1), #positive control (G3) vs therapy (G2), §negative control (G4) vs prophylaxis (G1), $therapy (G2) vs prophylaxis (G1).
Table 1 RPP data over time [MW (SD), *P,0.05 vs prophylaxis and positive control]. Black arrows indicate the time of HBOC-200 application RRP (mm Hg)
! Coronary artery occlusion
!
Reperfusion
Time (hh:mm)
Negative control
Positive control
Therapy
Prophylaxis
00 : 10 00 : 15 00 : 25 00 : 35
23.1 (2) 24.6 (3) 24.5 (2) 25.2 (2)
24.5 23.4 24.7 25.8
(3) (3) (3) (3)
23.0 23.1 23.5 23.7
(3) (3) (3) (3)
25.5 (6) 23.9 (6) 23.3 (7) 22.5 (7)
00 : 45 00 : 50 01 : 00 01 : 05
24.1 (2) 24.9 (2) 24.8 (3) 25.3 (3)
24.5 21.8 25.3 25.8
(5) (3) (4) (3)
23.8 19.2 22.3 20.4
(4) (5) (5) (4)
22.9 (7) 22.5 (6) 21.6 (5) 22.9 (6)
01 : 12 01 : 20 01 : 30 01 : 40 01 : 50 02 : 00 02 : 10 02 : 40 03 : 10 03 : 40 04 : 10 04 : 40 05 : 10
25.4 (4) 27.3 (3)* 25.7 (2) 24.9 (4) 24.9 (1) 24.4 (1) 25.1 (2) 24.8 (4) 25.6 (3) 25.4 (3) 25.1 (2) 25.5 (3) 23.9 (5)
23.7 23.6 22.9 23.7 24.4 24.3 24.9 25.5 24.0 24.5 24.2 24.9 23.8
(4) (3) (4) (3) (3) (3) (3) (3) (3) (3) (3) (3) (3)
19.8 22.1 20.3 21.6 21.6 22.1 21.5 21.4 21.4 21.7 20.1 22.4 22.5
(3) (4) (3) (4) (4) (4) (3) (3) (3) (2) (5) (3) (5)
21.1 (5) 21.4 (4) 22.1 (6) 22.2 (5) 22.6 (4) 22.6 (4) 23.1 (5) 24.2 (4) 24.2 (4) 22.6 (2) 21.7 (4) 24.1 (3) 23.6 (5)
MAP was observed immediately after prophylactic administration of HBOC-200 (G1) in comparison with all other groups (G2 –4). However, in contrast to results of the heart rate, this episode was only significant until 90 min before the end of experiment compared with the positive control group (G3). When HBOC-200 was administered in the therapy group (G2), a similar trend for MAP was noted; nevertheless, the difference did reach significance only 10 min after reperfusion in comparison with the positive control (G3). Although HBOC-treated groups (G1 and 2)
exhibited lower heart rates and elevated MAPs, the RPP of all ischaemic groups (G1– 3) were comparable over the time of experiment with the exception of RPP being higher in the negative control group compared with G1 and 2 once 15 min after reperfusion (Table 1).
Blood gas analysis and temperature There were no significant differences between the groups with respect to PCO2, PO2, pH, BE, Naþ, Kþ, and
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50
Haemoglobin solution reduces infarct size
Area at risk, infarct size, and areas of impaired perfusion (NR) The average risk area in the three ischaemic groups (G1–3) ranged between 20% and 25% of the left ventricle [G1, 21 (9)%; G2, 23 (8)%; G3, 25 (15)%]. The negative control group (G4) experienced slightly smaller risk areas [16 (5)%]. However, there were no significant differences between the groups with respect to the area at risk. After 30 min of coronary occlusion and 240 min of reperfusion, the area at risk that developed infarction was 48 (17)% in the positive control group (G3), whereas no infarction was detected by TTC staining in the negative control (G4) except minor zones adjacent to the suture. Administration of HBOC-200 in a prophylactic and therapeutic manner (G1 and 2) reduced infarct size significantly to about 25% of the area at risk [G1, 25 (13)%; G2, 22 (20)%, P,0.05]. The anatomic zone of areas of impaired perfusion was delineated by thioflavin-S staining. In positive control rabbits, 35 (12)% of the area at risk showed a perfusion defect (NR phenomenon). Minor perfusion defects in the negative control group (G4) corresponded again to the suture and averaged 5 (3)% of the area at risk (P,0.008 vs G3). The extent of areas of impaired perfusion as percentage of the area at risk was not significantly
Table 2 Blood gases and haemoglobin concentrations during experimental protocol. Data are mean (SD). PaCO2 and PaO2, arterial partial pressure of carbon dioxide and oxygen, respectively; Hbt, total haemoglobin concentration; MetHb, met-haemoglobin; HbO2, oxyhaemoglobin; Hbp, plasma haemoglobin. Appl., application; Occl., occlusion; Rep., reperfusion. P,0.05: *positive control vs prophylaxis, #positive control vs therapy, §negative control vs prophylaxis, $therapy vs prophylaxis, †negative control vs therapy Time (hh:mm)
Coronary artery occlusion 00 : 30 Baseline
PaCO2 (mm Hg) Prophylaxis Therapy Positive control Negative control PaO2 (mm Hg) Prophylaxis Therapy Positive control Negative control Hbt (g dl21) Prophylaxis Therapy Positive control Negative control MetHb (g dl21) Prophylaxis Therapy Positive control Negative control HbO2 (%) Prophylaxis Therapy Positive control Negative control HbP (g dl21) Prophylaxis Therapy Positive control Negative control
35 35 35 34
(6) (3) (5) (5)
00 : 45 Appl. 1110 min
41 36 35 34
(5) (3) (5) (5)
01 : 05 Occl. 15 min
37 35 35 34
(4) (3) (5) (5)
01 : 20 Appl. 2110 min
36 38 36 34
(2) (5) (6) (2)
01 : 32 Rep. 12 min
35 34 35 34
(2) (13) (5) (3)
05 : 30 Rep. 1240 min
38 39 38 41
(6) (4) (3) (13)
124 (28) 115 (25) 120 (19) 109 (33)
113 (27) 106 (23) 118 (24) 126 (33)
126 106 120 116
(25) (19) (22) (24)
166 (98) 141 (63) 180 (127) 111 (22)
147 (50) 104 (20) 109 (20) 113 (24)
142 (29) 166 (110) 138 (31) 162 (99)
13.2 (1.0) 13.0 (0.8) 12.9 (0.6) 12.2 (0.6)
12.5 (0.9) 12.1 (1.1) 11.9 (1.0) 11.7 (0.5)
12.5 12.3 12.0 12.0
(1.1) (0.8) (1.0) (0.5)
12.2 (0.9) 12.0 (0.4) 11.6 (0.9) 11.7 (0.5)
11.9 (0.9) 11.8 (0.7) 11.3 (1.0) 10.8 (1.0)
11.2 (0.9) 11.4 (1.2) 11.4 (0.7) 10.3 (1.1)
0.7 (0.1) 0.6 (0.1) 0.6 (0.2) 0.7 (0.1)
0.8 (0.3) 0.6 (0.1) 0.6 (0.2) 0.6 (0.1)
0.8 0.7 0.6 0.6
(0.2) (0.1) (0.2) (0.1)
0.8 (0.1) 0.7 (0.2) 0.6 (0.0) 0.6 (0.1)
0.7 (0.2) 0.7 (0.1) 0.8 (0.1) 0.6 (0.1)
96.2 (1.1) 95.4 (1.8) 96.0 (1.1) 95.5 (2.1)
94.4 (1.0) 95.0 (1.5) 95.7 (1.4) 97.1 (1.1)§
94.9 95.0 95.9 96.4
(1.3) (1.6) (1.2) (0.8)
95.4 (0.9)$ 92.6 (2.6) 95.1 (1.5) 96.1 (1.5)†
95.6 (1.6) 93.3 (2.9) 95.7 (1.3) 96.6 (1.1)†
1.2 (0.1) 0 (0)$ 0 (0)* 0 (0)§
1.2 0 0 0
(0.1) (0)$ (0)* (0)§
1.0 (0.1) 1.1 (0.1) 0 (0)*,# 0 (0)§,†
0.8 (0.1) 0.8 (0.1) 0 (0)*,# 0 (0)§,†
0 0 0 0
(0) (0) (0) (0)
501
0.8 (0.2) 0.8 (0.2)# 0.6 (0.1) 0.7 (0.1) 95.6 (0.9) 93.0 (6.1) 96.0 (1.2) 95.8 (1.5) 1.1 (0.1) 1.1 (0.1) 0 (0)*,# 0 (0)§,†
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glucose concentration (Table 2). The total haemoglobin and carboxyhaemoglobin concentrations did not differ between the groups during the whole observation period. HBOC-200 treated groups (G1 and 2) had methaemoglobin levels up to a peak concentration of 0.8 g dl21. However, the difference only reached significance in the therapeutic group (G2) in comparison with the positive control group (G3) after 20 min of coronary occlusion. The oxyhaemoglobin concentration in G4 was significantly higher in comparison with the prophylactic group (G1) after HBOC-200 application. During reperfusion, the oxyhaemoglobin concentration in the therapeutic group (G2) was significantly reduced in comparison with the negative control group (G4). At the beginning of reperfusion, the oxyhaemoglobin concentration in the prophylactic group (G1) was significantly higher in comparison with the therapy group (G2). The administration of HBOC-200 was accompanied by the expected elevated plasmatic haemoglobin concentration up to a maximum of 1.3 g dl21, followed by a moderate decrease in the prophylactic and the therapeutic groups (G1 and 2). The body core temperature was similar in all groups.
Rempf et al.
affected by therapeutic [G2, 34 (22)%] or prophylactic [G1, 26 (15)%] HBOC-200 treatment in comparison with the positive control group (G3).
Table 3 RMBF in the area at risk (blue dye negative areas) of the apex of the heart (ml min21 g21) at 25 min of coronary occlusion and 2 min of reperfusion Prophylaxis
Therapy
Positive control
Negative control
0.05 (0.04)
0.11 (0.07)
0.05 (0.02)
1.91 (0.7)
3.21 (1.36)
2.84 (0.56)
2.92 (1.41)
2.11 (0.54)
Regional myocardial blood flow During occlusion, all groups were equally ischaemic in the risk area with blood flow close to zero (Table 3). In all ischaemic groups, average blood flow in the previously ischaemic region increased after 2 min of reperfusion. No statistically significant difference between the groups could be observed.
Discussion
after reperfusion was significantly improved with HBOC-201 treatment in comparison with the saline control group. In the second model, called the myocardial infarction model, ischaemia was similarly created by LAD stenosis to 80– 90% flow reduction with concurrent cardiac pacing at a rate of 10% above spontaneous heart rate. Ischaemia was maintained for 195 min and reperfusion was allowed for 180 min. Dogs were treated during ischaemia at different times with different doses of HBOC-201 (0.5 and 1 g kg21 at 15 min ischaemia and 1 g kg21 at 60 min ischaemia). Again, no evidence of impaired coronary flow or significant coronary vasoconstriction was found in animals treated with HBOC-201. When 1 g kg21 HBOC-201 was given 15 min after the onset of ischaemia HBOC, an increase in LAD flow in comparison with the control groups was reported. The mean coronary resistance was lowered in HBOC-201-treated animals after 60 min of ischaemia in comparison with the saline control group, and myocardial infarct size as percentage of the area at risk was significantly reduced when 1 g kg21 HBOC-201 was given 15 min after ischaemia in comparison with the control groups. Furthermore, George and colleagues20 reported that infarct size in the heart layer most vulnerable to ischaemia was significantly diminished in animals with delayed, modest, and enlarged HBOC-201 treatment. One of our previous studies13 showed that prophylactic application of 0.4 g kg21 before acute LAD occlusion (25 min) and reperfusion (120 min) reduced infarct size, density of DNA single-strand breaks, and severity of cardiac arrhythmias compared with a saline-treated control group in rats. However, when given during LAD occlusion before reperfusion, HBOC-200 failed to show beneficial effects.13 Results of the present study show that both prophylactic and therapeutic treatment with HBOC-200 is effective to reduce myocardial infarct size, in comparison with the saline-treated positive control group after 30 min of CAO and 240 min of reperfusion in an animal model of low collateralization. Although reasons for these different results are unknown, the duration of reperfusion may have an impact (120 vs 240 min of reperfusion). In the present study, ischaemia and reperfusion were confirmed by a microsphere technique. All treated groups were equally ischaemic in comparison with the positive control and no animal showed a failure of the coronary artery reperfusion at 2 min of
502
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In a recent meta-analysis including all available clinical trials evaluating artificial oxygen carriers like HBOC-200, increased risk of mortality and myocardial infarction was found,7 18 and this led to a demand that any new or existing HBOC should be subjected to preclinical studies in animal models that replicate the known toxicities of HBOC demonstrated in humans before further clinical trials of this class of product are allowed to proceed. In contrast to the findings of the recent meta-analysis, experimental animal studies investigating the effects of HBOC-200 and HBOC-201 on infarct size were able to show that both HBOC solutions can reduce myocardial infarct size in animal models.13 19 20 Caswell and colleagues,19 in their study of HBOC-201 pre-ischaemic-treated dogs (10% of total blood volume was removed and replaced with HBOC-201), found a reduction in creatine kinase MB isoform release, a reduction in neutrophil infiltration in the area at risk, and a 56% reduction in myocardial infarct size in comparison with a saline-treated control group after 90 min of ischaemia and 270 min of reperfusion. Ischaemia was performed by complete left anterior descending (LAD) occlusion. RMBF from the epicardium and endocardium during baseline was comparable between HBOC and saline-treated dogs. At ischaemia, both groups showed a significant RMBF decrease in comparison with baseline values without significant differences between saline- and HBOC-201-treated animals. George and colleagues20 investigated in two distinct canine models the effects of HBOC-201 on LAD flow, regional myocardial function, RMBF, and myocardial infarct size. Ischaemia was produced by acute LAD stenosis to 80– 95% flow reduction from baseline. In the first model, called the regional ischaemia model, the stenosis was maintained for a total of 60 min followed by 15 min of reperfusion. After 15 min of ischaemia, dogs were treated with 1 g kg21 HBOC-201. Compared with a saline and phenylephrine control group, LAD flow improved and mean LAD resistance was lowered significantly in the HBOC-201-treated group. Additionally, RMBF in the area at risk was significantly higher with HBOC-201 treatment at the end of ischaemia and regional myocardial function
25 min coronary artery occlusion 2 min reperfusion
Haemoglobin solution reduces infarct size
after 240 min of reperfusion, suggesting that HBOC-200 has no beneficial effect on NR areas. It has been suggested that bovine haemoglobin given before ischaemia may reduce the severity of ischaemia by preloading the myocardium with oxygen or by aiding in oxygen delivery to hypoxic tissue.13 19 20 However, our recent data suggest that therapeutic treatment was as effective as the prophylactic approach to reduce myocardial infarct size in an animal model with poor collateralization. Thus, it appears that the mechanism of action also depends on the time of reperfusion. A potential mechanism for the reduction in infarct size after myocardial ischaemia could be improved oxygenation in the respective tissue. The enhanced oxygen off-loading capacity represents an important part of oxygen delivery responsible for tissue oxygenation by HBOC. HBOC solutions have a high oxygen diffusion coefficient in comparison with whole blood3 and also provide increased oxygen off-load from the red blood cells.4 The augmented oxygen extraction in the presence of HBOC provides enhanced oxygen supply at the tissue site. When compared with red blood cells,30 HBOC seems to have a higher oxygenation potency due to the plasmatic oxygen transport and enhanced oxygen release from HBOC and from adjacent erythrocytes. In this context, the damaged myocardial tissue seems to benefit from the oxygenation with HBOC-200 in the present model of myocardial ischaemia and reperfusion, which may result in a decreased infarct size by prophylactic and therapeutic treatment.
Conclusion We were able to show that prophylactic and therapeutic application of HBOC-200 reduces infarct size in a myocardial ischaemia and reperfusion model in rabbits. This reduction of infarct size is not accompanied by a reduction of areas of impaired perfusion. Therefore, clinical trials with a low dosage of HBOC to provide improved oxygen delivery in an organ of impaired perfusion could be of interest.
Funding This work was completely financed by the Department of Anaesthesiology, University Hospital Hamburg Eppendorf, Germany.
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503
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reperfusion. However, in the present study, we did confirm an alteration on haemodynamics by HBOC-200 treatment in our model of myocardial infarction and reperfusion. When HBOC-200 was given before CAO, a significant increase in MAP and a decrease in heart rate were observed. The effect of MAP was less obvious when HBOC-200 was given during ischaemia. Therefore, the observed infarct size reduction by HBOC-200 might be attributable to changes on myocardial oxygen consumption by haemodynamic parameters. However, the RPP for all ischaemic groups was only significantly different at one time after reperfusion. One limitation of our study is that, like in all other animal studies, rabbits with normal coronary arteries and without significant collateral coronary blood flow are not necessarily comparable with humans with significant coronary artery disease dependent on collateral flow to prevent extensive myocardial infarction. Another limitation could be in the limited time of observation of the animals. In many clinical trials, with the use of higher dosages of HBOC administered in the perioperative period, myocardial infarctions in humans were identified over a period of days instead of immediately after reperfusion. Results of a possible administration of a higher dosage of HBOC in our trial are also unknown. Additionally, the study is limited, in that we did not use another control group of non-oxygen carrying solution with similar colloid osmotic pressure, as the changes we detected probably may not directly be related to the oxygen carrying capacity of HBOC. As previously described, factors like a high colloid osmotic pressure resulting in a haemodilution by intravascular fluid shift may have an influence on our results, too.21 There is evidence from animal studies that the vasoconstrictive side-effects associated with HBOC treatment are caused by nitric oxide scavenging, endothelin release, or modulation of alpha-adrenoreceptors.22 – 24 This vasoconstrictive effect of cell-free haemoglobin solutions might be deleterious in circumstances of areas of impaired perfusion (NR) after myocardial ischaemia and reperfusion. It is known that a part of the myocardial capillary network after ischaemia is irreversibly closed at the time of reperfusion, despite restoration of epicardial vessel patency.25 The extent of resulting areas of NR depends on the length of ischaemia and the effects continue during reperfusion.11 This ‘NR phenomenon’ is known to be a consequence of a complex interaction of endothelial cell damage, vascular dysfunction, and perivascular swelling and also microembolism by platelets and leucocytes and their interactions with the endothelium.25 Studies in animals11 25 26 and clinical investigations27 28 demonstrated a close correlation between areas of impaired perfusion and myocardial necrosis. Furthermore, it has been described that NR predicts infarct size expansion.29 In the present study, the areas of NR in the area at risk were not significantly different from HBOC-200 treated and untreated hearts
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