BJA Advance Access originally published online on June 4, 2007
British Journal of Anaesthesia 2007 99(2):177-183; doi:10.1093/bja/aem116
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Effects of cardiopulmonary bypass on neurocognitive performance and cytokine release in old and diabetic rats
1 Division of Perioperative Care and Emergency Medicine, University Medical Center Utrecht, Mail Stop Q 04.2.313, PO Box 85500, 3508 GA Utrecht, The Netherlands
2 Klinik für Anaesthesiologie, Technische Universität München, Klinikum rechts der Isar, Ismaniger Strasse 22, 81675 Munich, Germany
* Corresponding author: Division of Perioperative Care and Emergency Medicine, University Medical Center Utrecht, Heidelberglaan 100, Mail stop Q 04.2.313, 3508 GA Utrecht, The Netherlands. E-mail: c.j.kalkman{at}azu.nl
Accepted for publication February 16, 2007.
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Abstract |
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Background: Age and diabetes mellitus have been identified as independent risk factors for cognitive decline after cardiac surgery with cardiopulmonary bypass (CPB). We tested the effects of CPB on cognitive function in aged and diabetic rats utilizing the Morris water maze (MWM).
Methods: Aged rats (26 months) were randomized into a sham group (cannulation but no CPB, n = 11) and a 90 min CPB group (n = 11). In addition, young rats (n = 14) were made diabetic with streptozotocin 9 weeks before experimentation and randomized to a sham or 90 min CPB group. Cytokine release [interleukin (IL-6)] and short-term MWM performance (days 8–14 after operation) were assessed in all animals. Long-term MWM performance (8 weeks after operation) was assessed in aged rats only.
Results: There were no differences between the aged groups in short-term (P = 0.58) or long-term MWM performances (P = 0.69). The diabetic animals also showed no differences between the sham and CPB groups in MWM performance (P = 0.64). IL-6 assays showed an increased inflammatory response after CPB in the diabetic animals, but not in the elderly groups.
Conclusions: Ninety minutes of normothermic CPB had no deleterious effect on neurocognitive outcome in elderly or chronically diabetic animals, suggesting that CPB in itself is not a sufficient stressor of the rat central nervous system.
Keywords: complications, neurological; heart, cardiopulmonary bypass; immune response; model, rat
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Introduction |
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Neurological and cognitive impairments after cardiac surgery are serious complications with major impacts.1 2 The use of cardiopulmonary bypass (CPB) is thought to be an important contributing factor. A reliable small animal CPB model would allow us to study the relative importance of different aetiological factors and provide a tool for efficient preclinical testing of putative neuroprotective strategies and compounds. Mackensen et al.3 and Grocott et al.4 were the first to describe a survival model for CPB in healthy young rats.
Several human studies have identified age and diabetes as independent risk factors for neurocognitive decline after cardiac surgery.2 5 Increased vulnerability of the ageing brain to various insults has been repeatedly demonstrated in experimental models of neurological injury.6–9 Also, experimentally induced diabetes in young rats causes irreversible cerebral microvascular changes, decreased cognitive performance and increased infarct volumes in experimental stroke models.10
The present study was designed to incorporate these clinically relevant risk factors into the rat model of CPB.3 4 We exposed aged and diabetic rats to 90 min of CPB and evaluated neurocognitive outcome and the inflammatory response. We hypothesized that 90 min of CPB would lead to cognitive impairment in the Morris water maze (MWM) in these at risk groups.
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Methods |
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The Utrecht University Animal Experimentation Committee approved all procedures and testing, according to the European Convention on Animal Care.
Experimental groups
Old rats
Thirty-eight male Wistar–Hannover rats (Harlan, Horst, The Netherlands), 25–27 months of age from a population with a median lifespan of 26 months, were randomized into two groups: (i) a group subjected to 90 min of CPB and (ii) a sham-operated group.
Diabetic rats
Male Wistar rats, weighing 400–450 g (n = 14), were injected 8–9 weeks before the start of the experiment with 33 mg kg–1 streptozotocin (STZ) i.v. (Serva, Heidelberg, Germany) to induce chronic diabetes mellitus,11 and randomized into CPB and sham groups. The diabetes was untreated. Rats were housed per two in Macrolon® cages with sawdust bedding. An 8 a.m. off–8 p.m. on lighting cycle was used. Food and water were available ad libitum.
Anaesthesia, surgical preparation, and CPB system
Anaesthesia was induced using isoflurane 5% in oxygen-enriched air. The animals were intubated with a 16 G catheter (Abbott, Hoofddorp, The Netherlands) used as tracheal tube and mechanically ventilated (FIO2, 40%; frequency, 30 min–1; peak pressure, 15 cm H2O) to achieve normocapnia (verified by mainstream capnography and arterial blood gas analysis). Atropine 0.05 mg was given subcutaneously to prevent excess pulmonary secretions. Anaesthesia was maintained with isoflurane 2.0–2.5%. The surgical procedure12 started with cannulation of the femoral artery with a 26 G catheter (Abbott, Hoofddorp, The Netherlands) for arterial pressure monitoring. Immediately thereafter, 150 IU of heparin and 5 µg of fentanyl were given. Glucose (Accu-Check Sensor 2, Roche, Almere, The Netherlands), blood gases (ABL 775 and OSM 3, Radiometer, Copenhagen, Denmark), and haematocrit (Hct) were analysed. The experiment was discontinued if the starting Hct was <0.34 to control for effects of health on behavioural performance.13 The tail artery was cannulated with a 20 G catheter for arterial inflow and a second dose of 150 IU of heparin was administered. A modified multi-orifice 4.5 French pediatric cannula (Desilets-Hoffman Catheter, Cook, Son, The Netherlands) was advanced through the right external jugular vein into the right atrium for venous outflow. The CPB circuit consisted of a newly designed 8 ml Plexiglas® venous reservoir, a roller pump (Verder, Haan, Germany), and a small-volume oxygenator (M.Humbs, Valley, Germany). The oxygenator has two Plexiglas® shells (12.8 cm x 12.8 cm x 2.7 cm) that carry a sterile, disposable three-layer artificial diffusion membrane, made from hollow polypropylene fibres (Jostra AG, Hirrlingen, Germany). The surface area available for gas exchange was 558 cm2. To prevent excessive heat loss, the oxygenator had an integrated heat exchanger. A glass-bubble trap was located between the arterial outflow of the oxygenator and the animal. All parts were connected through disposable silicone tubing. The circuit was primed with 8 ml of hydroxyethyl starch (HES) 6% and 4.5 ml of homologous-donor blood, obtained from sham-operated animals as described below. The CPB at a flow rate of 100–150 ml kg–1 min–1, adjusted to maximize flow and to maintain a minimal venous reservoir blood level, was carried out for 90 min. During CPB, HES was added as needed. Mean arterial pressure was maintained between 60 and 80 mm Hg, if necessary with the use of phenylephrine 10 µg ml–1, 0.1–1.0 ml h–1.
At the start of the CPB/sham period, pancuronium (0.2 mg kg–1) and fentanyl 5 µg were administered. During CPB ventilation was stopped and anaesthesia maintained with isoflurane added to the oxygenator gas flow (600 ml min–1 of O2 and 18 ml min–1 of CO2). Temperature was monitored using a thermistor placed underneath the left temporal muscle adjacent to the skull, and maintained at 37.5 (1.0°C) using an electrical heating pad and a heat-reflecting blanket.
After 90 min of normothermic bypass, ventilation was restarted (FIO2=1) and the extracorporeal circuit disconnected. The cannulae were removed and wounds closed. The animals remained ventilated for another 60 min, and recovered in an oxygen-enriched environment thereafter. Sham animals underwent the same cannulation procedures, except for connection to the extracorporeal circuit and discontinuation of ventilation. In addition, 9 ml of blood was replaced with HES 6% at the start of the sham period, to reach a similar Hct to that of the CPB animals.
Neurocognitive testing
Old animals
The investigator performing the tests was blinded to treatment allocation. The animals underwent neurocognitive testing in our MWM set-up,12 a 2 m diameter black pool with a fixed hidden platform submerged 1 cm below the water surface in one quadrant and various visual cues on the surrounding walls. The rat could climb on the platform to escape from the necessity to swim. Testing was performed 1 h after the start of the darkening period in a red-lighted room and consisted of three trials daily. During each trial the animals were placed in the pool at a randomly assigned, different quadrant. Trials were limited to a 120 s search period, after which the animal was placed on the platform. A computerized video tracking system (Ethovision v1.9, Noldus, Wageningen, The Netherlands) measured latency (time to reach the platform), swimming speed, and overall distance. Testing for short-term effects was started at the eighth postoperative day and continued for 7 days to allow a latency floor value to be reached. Long-term effects were assessed in a reversed MWM set-up during a 4-day period starting at 8 weeks after operation. In the reversed set-up, the platform was located in a quadrant other than that used in the short-term set-up and cues were changed.
Diabetic animals
Diabetic animals started neurocognitive testing at the fourth postoperative day and continued testing for 5 days, at which time a latency floor value was expected to be reached. No long-term testing was performed because of the poor physical condition of the animals as a result of untreated diabetes.
IL-6 analysis
The experimental protocol for interleukin-6 (IL-6) determinations was the same for old and diabetic animals. Blood samples from each animal were obtained just before the start of CPB, after 90 min of CPB, and 1 h after the end of CPB. A total of 0.5 ml of blood was taken for each sample; plasma was separated in EDTA-tubes (Microtainer, BD, Alphen aan den Rijn, The Netherlands) and stored at –80°C. Duplicate analysis of plasma IL-6 levels was performed by an investigator blinded to group allocation. Assays were performed using rat-specific ELISA kits (Quantikine M IL-6 Immuno assay; R&D Systems, Minneapolis, MN, USA).
Statistical analysis
Data from cognitive tests are presented as mean (SD) and analysed by repeated measurements ANOVA, with Greenhouse–Geisser correction if appropriate. Data from cytokine assays are presented as medians with minimum/maximum. Differences between group cytokine levels were assessed using the Mann–Whitney U-test. Physiological values were analysed using Student's t-test. P-values <0.05 were considered significant. An estimation of the number of animals required per group was based on group sizes and differences found in earlier studies of Mackensen and colleagues,3 Dieleman and colleagues,12 and Ma and colleagues.14 Although in these studies an oversized oxygenator was used, this did not influence our a priori power analysis, as the exposure of blood to the extracorporeal system is thought to play a more important role than oxygenator surface per kilogram the animal is being exposed to.
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Old animals
Of the initial 38 aged rats, only 22 (n = 11 per group) were actually used in the experiment. The others had to be excluded because of: death during transportation and housing (n = 1), visible masses (n = 7), and low-body weight or Hct <0.34 after the first analysis (n = 8). Two animals in the CPB group died within the first postoperative hour; one because of catheter-related problems (cardiac tamponade), and the other of unknown causes.
Table 1 shows physiological values during the experiment. During CPB, temperatures were lower in the CPB group when compared with the sham group. At 1 h after operation, PaO2 was also lower (but >100 mm Hg) in the CPB group.
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Diabetic animals
The weight of the animal decreased from around 400 g at the time of the STZ injection to 370 g 9 weeks thereafter, because of severe metabolic dysfunction. All animals survived the surgery and completed the study (n = 7 for each group). Perioperative physiological values in the diabetic groups are listed in Table 2. At the start of the experiment, all blood glucose levels were >17 mmol litre–1. Mean arterial pressure was somewhat lower in the CPB group, but still within the normal range. At the end of CPB, temperatures were lower in the CPB group. In addition, during the postoperative period, PaO2 was lower (but >13.3 kPa) in the CPB animals.
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Neurocognitive tests
Old animals
Figure 1A shows MWM performance (latency) of old animals. In each group, there was improvement over time (P < 0.001) as evidence of learning. A latency floor was reached on day 13 after operation, implying no further learning. There was no difference between the study groups (P = 0.58). The reverse MWM at 8 weeks also showed improvement over time (P = 0.024), but again without differences between the groups (P = 0.69). There were no significant differences in swimming speed, or in distance swam (data not shown).
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Diabetic animals
In diabetic animals, both the CPB and sham groups showed significant learning over time (P < 0.001), but there were no differences between the groups (P = 0.64). Latencies did not improve further after 4 days of testing, reaching a floor at an average of 32 s on day 8 after operation (Fig. 1B).
IL-6 analysis
IL-6 levels are shown separately for the old and diabetic groups (Fig. 2A and B).
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In the elderly animals, IL-6 concentrations were not different between the two groups at any time point. In both sham and CPB animals, the values at 90 min CPB and 1 h post-CPB were higher than baseline (P = 0.02 and 0.001 for the sham animals, P = 0.01 and 0.003 for the CPB animals).
Baseline values in diabetic animals were not statistically different (P = 0.45) between the sham and the CPB group, but at 90 min CPB and 1 h post-CPB IL-6 levels in the CPB group were significantly higher than in the sham group (P = 0.002 and 0.005, respectively). In addition, a time-effect was noted in the CPB group: the IL-6 levels were higher at 90 CPB and 1 h post-CPB time points when compared with baseline (P = 0.003 for both time points). In the sham group, only the 1 h post-CPB value was different from the baseline (P = 0.03).
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Discussion |
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In the present studies, we subjected rats with increased vulnerability for central nervous system (CNS) dysfunction (aged and diabetic animals) to 90 min of normothermic CPB and evaluated short- and long-term neurocognitive outcome in the MWM. Compared with sham-operated aged or diabetic animals, there was no evidence of impaired visuo-spatial learning in animals exposed to 90 min of normothermic CPB. In the aged animals, plasma levels of the pro-inflammatory cytokine IL-6 increased during CPB and in the post-CPB period, but were comparable between CPB and sham animals. However, in the diabetic animals, CPB did cause a significant increase in IL-6 when compared with sham-operated animals.
The results of this study are in accordance with our previous study,12 in which we found that CPB in young healthy animals did not lead to cognitive impairment in the MWM. We stated that using young healthy animals in a model for a condition that mainly affects the elderly was not appropriate. We hypothesized that by using animals with increased susceptibility for neurcognitive deficits, CPB would induce cognitive impairment.
We opted to use old animals because of their well-known susceptibility to CNS injury. Not only do aged rats demonstrate increased infarct volumes in stroke models,8 9 they also have a reduced capacity for neurogenesis after ischaemia15 and increased apoptosis and impaired MWM performance after intermittent episodes of hypoxia.6 Reduced capacity for regeneration and loss of neurons and synapses, especially in the hippocampus, leads to deficits in spatial learning and memory.16 17
Treating rats with STZ produces an established animal model for diabetes.11 In this model, it has been shown that 8 weeks of untreated STZ-induced diabetes produces functional and cognitive deficits.18 19 We therefore chose to use animals from this model for diabetes as a second group with increased vulnerability for neurocognitive deficits. As expected, all animals in our study suffered from severe, irreversible hyperglycaemia after treatment with STZ. Because their hyperglycaemia was present for more than 9 weeks, all the diabetic complications of this model would have been present in our animals.
As in our previous study with this model, we used the MWM to assess cognitive function in our animals. The MWM is a well established and widely used spatial memory test for rats and mice, in which functional hippocampal integrity is essential for normal performance.20 Also, the MWM has repeatedly been shown to be sufficiently sensitive to detect deficits in spatial memory in aged rats in particular.6 17 20 It is therefore unlikely that we would have missed any difference in cognitive performance because our test was insensitive.
As these results apparently contradict two previous studies using this model, further discussion is warranted. Mackensen and colleagues3 reported a significant difference in MWM latency between young rats subjected to 60 min CPB and sham groups at days 3–12 after operation. Using the same experimental set-up, Ma and colleagues14 reported that rats exposed to xenon during CPB had lower MWM latencies on the first 2 days of testing. Both experiments ended at day 12 after operation and no long-term neurocognitive outcomes were studied. An important difference between the present study and this previous work, including our own,12 is that we now used a much smaller oxygenator. Until very recently, no miniature oxygenators were commercially available, and therefore the smallest neonatal oxygenator had to be used. The membrane surface of that oxygenator was approximately 20 times overdimensioned when compared with the clinical situation (0.66 vs. 0.02–0.04 m2 kg–1). For this study, we used a recently developed small animal oxygenator with a surface area of 0.09 m2 kg–1 in these rats. As a result, both priming volume and the blood–membrane contact could be reduced. Also, in the previous studies by other groups, the oxygenator was cleaned, sterilized, and reused, whereas we now were able to use a new membrane for each animal. It is conceivable that, despite rinsing, the oxygenator fibres in those earlier studies were still partly coated with protein residues, which might have augmented the inflammatory response. In contrast, in our own study using a large non-reused oxygenator, we were unable to demonstrate either cognitive deficit or an increased inflammatory response in the animals subjected to CPB. It is possible that the reuse of the oxygenator as done by the other groups may have contributed to both the observed impaired cognitive outcomes and the differences in activation of the immune system.
Despite the use of an appropriately sized oxygenator, we still needed to add 4.5 ml of donor blood to maintain Hct >0.28. In the sham animals, we diluted to the same Hct as the CPB animals. By combining these factors, analysis of inflammatory markers becomes more relevant. To investigate the effect of CPB on inflammation, we chose to measure the pro-inflammatory cytokine IL-6 because it is released relatively late in the inflammatory cascade. IL-6 levels are also consistently increased after cardiac surgery.21 Indeed, in diabetic rats IL-6 levels were significantly higher in the CPB group than in the sham animals. The lack of a between-group difference in the old animals could be attributed to the larger variability in the data (Fig. 2A). As a result of inter-individual differences in the ‘speed’ of the ageing process in rats, the variation in the elderly population is larger than in young animals. Also, many of these animals may still have had underlying conditions that influenced their cytokine response. In contrast, the diabetic animals were part of a more homogenous group, all with the same underlying illness for the same period of time. As expected, exposure to CPB increased IL-6 levels in these animals.
An important limitation of this study is the variation among old animals. Although we had anticipated this increased variation and increased our group size, because of the very high prevalence of visible masses and low preoperative Hct values, the number of excluded animals exceeded our estimates and 11 animals per group were included. We decided not to add animals to the study at a later point (from a different batch), because this is methodologically incorrect. This is even more so in studies where neurocognitive performance is the primary outcome measure, as animals from a different population would inevitably have different previous cognitive ‘experiences’, and therefore have non-comparable responses. A sample size of 12 would still have allowed an increase of 50% in MWM latency to be detected. For the diabetic animals a post hoc power analysis on day 2 shows that to detect a 45% increase in latency in this study with 80% power, nine animals are needed. Because the noted differences among the animals are minimal, we believe that the group sizes as chosen would have allowed us to detect apparent cognitive decline in these animals. The risk of having encountered a type II error in this experiment is thus relatively low. Other limitations to our study include the relatively short time-span we used to measure IL-6. This was chosen deliberately so we did not influence our cognitive test results by stressful blood sampling in awake animals. As a result, only the trend is measured and we may have missed peak levels. However, in this rat model, peak IL-6 levels occur between 2 and 4 h after start of 60 min CPB (unpublished observation) and therefore the time point 1 h after 90 min of CPB would still be sufficient to detect different rates of IL-6 release.
In conclusion, we were unable to demonstrate that exposing old or diabetic rats to 90 min of normothermic CPB caused persistent changes in neurocognitive function as measured by MWM performance. In the present model of CPB, using an appropriately sized, non-reused extracorporeal circuit—CPB alone is not a sufficient stressor of the CNS, not even in animals with increased susceptibility for CNS injury. These results are in agreement with recent clinical observations suggesting that avoidance of CPB using off-pump coronary artery bypass techniques has only limited effects on improving cognitive outcomes in humans.22–24 Nonetheless, the current rat CPB model may prove to be an excellent starting point to investigate the additive effects of CPB on perioperative (embolic) neurological injury during cardiac surgery.
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The authors would like to thank Dr Rogier Donders, Kees Brandt, Gijsbertjan van Breukelen, Sergej de Vries, and Jan-Dirk van Tiggelen for their valuable contributions to this study.
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