Although overall morbidity and mortality after cardiac surgery and cardiopulmonary bypass (CPB) have been improved over the last 50 yr, neurologic and neurocognitive deficits are common and significant complications.1–4 The difficulties in preventing or treating these deficits are at least in part based on the limitations to clearly understand the underlying mechanisms and etiologies. Investigating and defining these mechanisms might be more feasible in a nonclinical environment with free access to brain tissue, using a recovery model of CPB in the rat.5–7
The etiology leading to neurocognitive dysfunction after cardiac surgery is most likely multifactorial, and a systemic inflammatory reaction is perhaps one of the contributing factors.8,9 Systemic inflammation has been shown to be associated with an accentuated cerebral inflammatory response that is thought to contribute to neurocognitive deficits, although the link between cerebral inflammation and neurocognitive outcome has not been specifically studied. Human studies showed that the systemic inflammatory response can be reduced by minimizing the foreign surface of CPB or by off-pump procedures.10–12 However, these studies have not assessed neurologic or neurocognitive outcome. One human study comparing on-pump and off-pump procedures in regard to neurologic and neurocognitive outcome was unable to detect any difference between the two regimens in low-risk patients.13
The current experiment was designed to study perioperative systemic interleukin-6 (IL-6) concentrations as well as cerebral expression of nuclear factor-kappa B (NF-κB), a key transcription factor upregulated during cerebral ischemia and responsible for activating further proinflammatory variables such as cyclooxygenase-2 (COX-2), thus potentially influencing neurocognitive outcome after CPB in rats.14 To evaluate the impact of CPB circuit volume and foreign surface area on these outcomes, an appropriately sized small-volume rat oxygenator was compared with an oversized neonate oxygenator.
The following experimental protocols were approved by the institutional animal care committee, and all procedures described herein met the guidelines of the National Institutes of Health for animal care (http://www.nap.edu/readingroom/books/labrats/).Male Sprague-Dawley rats (21 days old) from Charles River Laboratories (Kisslegg, Germany) were housed under standard laboratory conditions (12 h light/12 h dark, lights on at 12:30 am, 22°C, 60% humidity, and free access to water and standard rat chow) 4 wk before the experiments for habituation to the changed day-night rhythm. This changed day-night rhythm allows the assessment of the rats' cognitive performance and behavior from 10:00 am until 12:00 pm, the time window closest to the night of the nocturnal rats (lights off at 12:30 pm). After a training period with the modified hole-board test, nonfasted rats (now about 9 wk old) were subjected to surgical preparation.
Four groups of randomly assigned rats were investigated. Control animals were neither anesthetized nor cannulated (n = 7). Sham-operated rats were anesthetized and surgically prepared but not exposed to CPB (n = 10). Another 20 rats were subjected to 90 min of complete CPB and were subdivided in 2 groups: animals in the CPB/rat oxygenator group were connected to an extracorporal circulation using an oxygenator that was specifically designed for rats (n = 10); animals in the CPB/neonate oxygenator group were connected to an oversized neonate oxygenator (n = 10).
Surgical Preparation and CPB
Rats weighing 352 ± 24 g were anesthetized with 3% isoflurane in oxygen in a plastic box. After orotracheal intubation using a 14-gauge cannula, the lungs were mechanically ventilated (45% O2/balance N2). Ventilation was adjusted to maintain a Paco2 of 35-45 mm Hg. During subsequent surgical preparation, anesthesia was maintained with 2.0%-2.5% isoflurane. Surgery was performed with an aseptic technique, and all surgical fields were subsequently infiltrated with 2% lidocaine. Between experiments, all reusable components were gas sterilized with formaldehyde to ensure sterility. For each animal, a new gas-sterilized oxygenator was used.
Pericranial temperature was monitored via a needle probe inserted into the right temporal muscle and servo-regulated at 37.5°C ± 0.1°C, which presents the normal rat's temperature (HYP-1 Newport, Santa Ana, CA) using a heating blanket and convective forced-air heating system. The tail artery was cannulated with a 20-gauge IV catheter, which later served as the inflow from the CPB circuit. After placement of the arterial catheter, rats were given 150 IU of heparin and 5 μg of fentanyl. Mean arterial blood pressure was monitored via the right superficial caudal epigastric artery, which was cannulated with PE-10 tubing. A 4.5F multiorifice cannula was inserted into the right external jugular vein through a neck incision. With the tip of this cannula at the junction of the inferior vena cava and the right atrium, optimal drainage of venous blood was accomplished. The proper location of the tip of the venous catheter was based on data from previous experiments using transesophageal echocardiography.15 The CPB circuit consisted of a venous reservoir, a peristaltic pump, a membrane oxygenator, and an arterial inflow cannula, all of which were connected via 1.6-mm internal diameter plastic tubing (Tygon®, Cole-Parmer Instrument Co., Vernon Hills, IL). Blood returning through the venous cannula to the venous reservoir (consisting of Plexiglas®, Evonik, Essen, Germany) was drained to the peristaltic pump (Masterflex®, Cole-Parmer Instrument Co.). Blood was pumped through an oxygenator and then returned to the animal via the arterial inflow cannula. An in-line flowprobe (2N806 flowprobe and T208 volume flowmeter, Transonics Systems, Ithaca, NY) was used to continuously measure CPB flow. No additional fluids were infused during CPB (besides drugs). The venous reservoir was placed 6-7 cm below the rat's level. When the venous drainage was insufficient during CPB, the venous catheter was repositioned. If repositioning failed to improve venous drainage, the venous reservoir was lowered further until a sufficient venous drainage was accomplished. Routinely, these measures would yield sufficient venous drainage throughout the CPB time.
Arterial blood gases were measured using a Rapidlab 860 blood gas analyzer (Bayer Vital GmbH, Fernwald, Germany). Baseline physiologic measurements, including mean arterial blood pressure, pericranial temperature, and blood gases, were performed 10 min before commencement of CPB. During CPB, anesthesia was maintained with 0.8%-1.2% isoflurane, subsequent doses of 5 μg of fentanyl as required, and 1.6 mg/h cisatracurium per infusion. All animals had the same surgical preparation and were exposed to the same anesthetic conditions as described above. The rats were then randomly assigned to the CPB/rat oxygenator or the CPB/neonate oxygenator group depending on the oxygenator used (described below) (n = 10) or served as sham-operated controls (n = 10). After preparation, rats in the CPB groups were subjected to 90 min of normothermic, nonpulsatile CPB with flow rates of 160-180 mL·min−1·kg−1 (normal cardiac output in awake rats). For this entire period, ventilation of the lungs was terminated. After 90 min of CPB, the animals were weaned from CPB without the need for inotropes or vasopressors. The heparin anticoagulation was allowed to dissipate spontaneously without supplemental administration of protamine. To avoid a decrease of serum glucose concentration below 80 mg/dL, glucose 50% was administered as required. After decannulation, rats remained anesthetized with 1.5%-2% isoflurane (without any additional fentanyl or cisatracurium), were intubated, and ventilated for 2 h. When the animals resumed spontaneous ventilation, the tracheas were extubated. The animals were allowed to recover in an oxygen-enriched environment for 2-4 h with free access to water and food. Thereafter, they were returned to the hole-board cages and housed in their familiar groups. Animals in the sham-operated group had all CPB catheters left in place for 90 min (thereby mimicking the surgical procedures for CPB), but they were not connected to the external CPB circuit itself. The animals were decannulated and recovered in the same fashion as the animals in the CPB groups.
Control animals were placed in regular cages and transported to the experimental laboratory where they were kept untreated for the same time as the sham and CPB animals.
For the assessment of long-term outcome, animals were allowed to survive for 21 postoperative days and underwent daily testing in the modified hole board. On the 21st day, the animals were killed in deep isoflurane anesthesia, and the brains were removed and stored at −80°C.
Oxygenator and Extracorporal Circuit
The small-volume rat oxygenator is built of two Plexiglas shells (12.8 × 12.8 × 2.7 cm) carrying the diffusion membrane (Fig. 1). The membrane consists of 3 layers of polypropylene hollow fiber mats (Jostra AG, Hirrlingen, Germany) glued together in a crosswise manner in order to improve oxygenation. The gas exchange area is 558 cm2. The prime volume of the oxygenator itself is 4 mL and the whole CPB circuit encompasses 10 mL. An integrated waterbath allows heating of the circuit and helps avoid a decrease in the rat's body temperature throughout extracorporal circulation. The CPB circuit was primed with 8 mL of whole blood of 1 heparinized donor rat, 2 mL of 6% hetastarch, and 100 IU of heparin.
In the CPB/neonate oxygenator group, a modified Cobe Micro® neonatal oxygenator (Cobe Cardiovascular, Arvada, CO) with a surface area of 0.33 m2 and a prime volume of 32 mL was used instead of the new rat oxygenator. The CPB circuit was primed with approximately 34 mL of whole blood of 2 heparinized donor rats, 4 mL of hetastarch, 150 IU of heparin, and 42 mg of bicarbonate to avoid acidosis.
Determination of IL-6 Concentration
To determine the systemic IL-6 concentrations after CPB or sham operation, blood samples were collected at 3 times: before CPB (pre-CPB), at 90 min of CPB (CPB 90 min), and 2 h after cessation of CPB (post-CPB 2 h), or at the equivalent times in the sham-operated group. Blood was immediately centrifuged (3000 rpm for 3 min), and serum was stored at −80°C. The cytokine IL-6 concentrations were measured by enzyme-linked immunosorbent assay. Rat-specific enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, MN) were used according to the manufacturer's instructions. For IL-6, the minimal detectable concentration was 10 pg/mL.
Modified Hole-Board Test
Cognitive performance and behavior were evaluated using the modified hole-board test. The modified hole board represents a custom-made combination of a hole board, originally designed to investigate cognitive, exploratory, and motivational variables in rats, and an open field, a test paradigm standardized to evaluate locomotor activity.16
The rats were housed in the hole-board environment that was divided into a test arena (40 × 60 × 50 cm) and 3 home cages (38 × 19 × 50 cm). The home cages and the test arena were separated by a transparent partition, perforated with holes to allow visual and olfactory contact between group mates. The hole board (20 × 40 cm) was placed in the middle of the test arena, thus representing the central area of an open field. Fifteen holes covered by lids, which could easily be opened, were staggered on the board. After opening, coil springs forced the lids back to their original positions. The lids of 3 holes contained puffed rice and were marked with white tape. All holes were flavored with the aroma of black currants to obscure the odor and smell of the puffed rice.
After habituation to the testing environment, rats learned the test procedure for 10 consecutive days (training phase). All animals were tested with marked and baited holes for 3 trials daily. The sequence of marked holes was randomly changed every day. After the training period, the rats' baseline conditions were obtained for 3 additional days. On the following day, the animals had surgical preparation with or without CPB. After surgery, the animals were returned to their home cages where they were tested daily for 21 days.
Several cognitive functions were assessed17: 1) deficit in overall cognitive performance was assumed if the time needed to complete 1 trial was extended; 2) deficit within the visuospatial long-term memory was assumed if rats visited nonbaited holes referred to as wrong choice or did not visit baited holes referred to as omission error; and 3) deficit within the visuospatial short-term memory was assumed if rats revisited a baited hole referred to as repeated choice. In addition to the assessment of the cognitive variables, the modified hole board allows evaluation of the following 5 patterns of behavior16: 1) latency to first entry on the board, board entries, immobility, and time on the board as indicators of avoidance behavior, i.e., anxiety; 2) the exploratory motivation, specified in directed exploration, assessed by the latency to the first hole visit, the total number of holes visited, and general exploration, assessed by the number of rearings; 3) the time spent grooming and the number of fecal and urine boluses as indicators for physiologic arousal; 4) the percentage of time spent in group contacts as an indicator for social affinity; and 5) the number of line crossings as a variable for locomotor activity.
Neurologic outcome was assessed daily with a neurologic score that was described previously.18 Briefly, we evaluated consciousness (normal [score 0] to lethargic [score 2]), grooming (normal [score 0] to none [score 2]), ability to walk (normal [score 0] to none [score 3]) and climb (climbs 90° [score 0] to no grip [score 4]), and force on a rotating grid (grips at 180° >5 s [score 0] to fall from the grid [score 4]). The scores were added to obtain a score for an individual rat.
After completion of the neurocognitive testing on the final day (21st postoperative day), animals were anesthetized with 5% isoflurane, and the brains were removed and frozen in tissue-freezing medium (Leica Instruments GmbH, Nussloch, Germany) using methylbutan, and then stored at −80°C.
Immunohistochemistry and Histology
Frozen brains were cut into 10-μm slices at bregma −3.3 mm using a cryotom (Microtom-Kryostat, HM 500 QM, Microtom GmbH, Walldorf, Germany). Adjacent slices were subjected to standard histology and immunohistochemistry. To detect NF-κB-positive neurons in the hippocampus, an immunohistochemical double staining was performed using polyclonal rabbit anti-NF-κB p65 (Abcam, Cambridge, UK) and monoclonal mouse anti-NeuN (Chemicon, Billerica, MA) against the neuronal structural protein N (NeuN). NF-κB-positive neurons in the hippocampus were counted under a light microscope (×400).
For standard histology, slices were stained with hematoxylin and eosin. To assess neuronal damage, 5 high-magnification fields (440-fold) in each of 4 brain regions (striatum, motor cortex, cingulate cortex, and hippocampus) were viewed by an investigator blinded to group assignment.
Physiologic, neurologic, cognitive, and behavioral values were analyzed using general linear models with the between-groups factor group, the within-group factor time, and their interaction terms. Effects of time levels were analyzed quadratically (time2), focusing on biphasic changes of variables during the observation period. Once time2 × group was significant, values were analyzed post hoc using factorial analysis of variance (ANOVA) followed by Tamhane's test.
Because the systemic IL-6 concentrations were distributed log normally, they were logarithmized before being subjected to statistical analysis as described above (general linear models and post hoc factorial ANOVA followed by Tamhane's test). Figure 2 displays the geometric means plus geometric sd, whereas in the Result section, the values are given as median (10%-90% percentile). The number of NF-κB-positive neurons in the hippocampus was analyzed using 1-way ANOVA with post hoc Tamhane's test.
All variables are presented as mean ± sd; P < 0.05. Statistical analyses were performed using SPSS 11.5.1 for Windows (SPSS, Chicago, IL).
Table 1 displays physiologic values from both CPB and sham-operated groups. The CPB/rat oxygenator group required a higher inspiratory oxygen concentration (Fio2 = 0.8) than the CPB/neonate oxygenator group (Fio2 = 0.5) to achieve comparable Pao2 values in both CPB groups. To maintain perioperative normoglycemia, IV glucose was administered in 4 animals in the sham-operated group (144 ± 72 mg), 3 in the CPB/neonate oxygenator group (100 ± 89 mg), and 3 in the CPB/rat oxygenator group (67 ± 29 mg).
There was 1 death in the CPB/neonate oxygenator group caused by insufficient venous return during CPB. An additional 3 animals died because of vascular perforation during cannulation of the jugular vein before group assignment. These 4 animals were replaced to keep sample size equal.
Figure 2 shows the time course for systemic IL-6 concentrations. The following had similar cytokine levels at baseline (sham operated: 1 pg/mL [1-1]; CPB/rat oxygenator: 1 pg/mL [1-16]; and CPB/neonate oxygenator: 1 pg/mL [1-1]). IL-6 concentrations increased significantly in the 2 CPB groups compared with sham-operated animals at 90 min CPB (sham operated: 1 pg/mL [1-1]; CPB/rat oxygenator: 703 pg/mL [372-5966]; and CPB/neonate oxygenator: 5136 pg/mL [1005-9209]) and at 2 h after CPB (P < 0.05). At 2 h after CPB, using the larger-volume neonate oxygenator caused a substantial increase in the magnitude of the IL-6 release compared with the rat oxygenator group (CPB/rat oxygenator: 220 pg/mL [16-415] and CPB/neonate oxygenator: 1400 pg/mL [592-5812]) (P < 0.05).
During the entire testing period, overall cognitive performance as well as long-term and short-term memory were not impaired and did not differ among groups (Fig. 3). Behavioral variables such as motivation and anxiety were not affected in any group (data not shown). Neurologic outcome was normal for all groups (data not shown).
The number of NF-κB-positive neurons in the hippocampus was increased in all 3 experimental groups compared with untreated controls (P < 0.05). Rats in both CPB groups demonstrated more NF-κB-positive neurons (271 ± 57 in the CPB/neonate oxygenator group and 269 ± 72 in the CPB/rat oxygenator group) compared with the sham-operated animals (173 ± 24) (P < 0.05), with no difference between the 2 CPB groups (Figs. 4 and 5). None of the animals showed any histologic damage in the hematoxylin and eosin staining.
This study showed higher levels of systemic IL-6 up to 2 h after CPB and increased expression of NF-κB in hippocampal neurons 21 days after CPB in rats compared with sham-operated controls. Although type and size of the oxygenator affected systemic IL-6 concentrations up to 2 h after CPB, hippocampal NF-κB expression was comparable on postoperative day 21. However, a systemic inflammatory response paired with cerebral inflammation was not accompanied by any impairment in histological, neurocognitive, or behavioral outcome.
The inflammatory cascade is specifically activated during CPB via several pathways. These include contact activation by the foreign surfaces of the circuit itself, the effect of ischemia-reperfusion injury, and endotoxemia.19,20 Direct evidence for an association of the systemic inflammatory response and neurocognitive dysfunction in cardiac surgery patients is lacking, even though the inflammatory reaction has always been considered as one potential etiologic factor for impaired neurocognitive outcome. In an attempt to explore this association, Westaby et al.21 reported, in humans, no link between systemic inflammation and cognitive dysfunction at 5 days and 3 mo after coronary artery bypass graft surgery in a small study. On the contrary, Ramlawi et al.22 showed that the inflammatory response 6 h and 4 days after cardiac surgery was associated with neurocognitive decline. One major advantage of an animal model is the availability of brain tissue to assess inflammation at the target, the brain itself. By doing so, Hindman et al.23 demonstrated that an increased IL-6 concentration 4 h after CPB in rats is associated with increased COX-2 expression in the brain. However, neuropsychological outcomes were not assessed. With this study, we have assessed all three aspects: systemic cytokine release, cerebral expression of NF-κB, and neurocognitive outcome. NF-κB was chosen as a marker of cerebral inflammation for several reasons. First, NF-κB is a ubiquitously expressed transcription factor that regulates expression of genes involved in inflammation, cell survival, and apoptosis and has been considered a promising molecular target to ameliorate cerebral injury.24,25 Second, NF-κB is upregulated during cerebral ischemia and has been shown to upregulate other proinflammatory variables such as COX-2.14,26,27 Third, the neuroprotective effect of hypothermia on cerebral injury has been speculated to be at least in part related to NF-κB inhibition,28 and fourth, NF-κB activation is also a crucial step in the signal transduction pathway underlying cerebral preconditioning.29
Of particular interest is the finding that although CPB caused increased systemic levels of IL-6 and higher numbers of NF-κB-positive neurons in the hippocampus, it did not lead to neurocognitive impairment. This is in agreement with human studies demonstrating a decreased systemic inflammatory response after off-pump procedures.30,31 However, other studies comparing off-pump and on-pump surgery were not able to detect any difference in neurocognitive outcome.13,32 Thus, neurocognitive impairment as observed after cardiac surgery may develop independent of the use of extracorporal circulation and other factors such as preexisting comorbidities, aortic manipulation, surgery, or hemodynamic compromise, which may be responsible for its occurrence. Of note, the type and size of the oxygenator affected only the systemic IL-6 concentration and not cerebral NF-κB expression. This might have been attributable in part to the fact that the extent of the foreign surface area is just a minor factor leading to transiently altered systemic inflammation but does not affect long-term cerebral inflammation. Considering all of these results, CPB in young, healthy rats does not seem to cause any neurocognitive deficits even though a systemic and cerebral inflammatory reaction is activated. This inflammatory reaction might be interpreted as an adaptive response to a nonphysiologic state that is not injurious enough in the absence of comorbidities or additional insults to cause neurocognitive impairment. Despite its documented effects on inflammation, NF-κB has frequently been associated with cell survival and antiapoptotic actions.33
Using this recovery model of CPB in the rat, studies revealed inconsistent results with regard to neurocognitive outcome and a systemic inflammatory response. Whereas some studies34,35 showed an increased IL-6 concentration after CPB compared with sham-operated animals, others showed comparable IL-6 levels for animals subjected to CPB or sham operation, even when such studies were performed in the same laboratory.7 Neurocognitive deficits after CPB in otherwise healthy rats were found in some studies5,36 but have not been confirmed by published works.7,35 Considering these and the current study, some key differences can be found in the oxygenator used (neonate or rat oxygenator) and in the learning test used for neurocognitive assessment (Morris water maze in previously published studies, and the modified hole-board test in the current study). The prime volume of the rat oxygenator is only one-eighth of the prime volume of the neonate oxygenator whereas the surface area is reduced sixfold. Thus, the smaller rat oxygenator better matches the clinical setting in that the relationship between prime and blood volume is comparable (10 mL prime versus 25 mL blood volume in the rat and up to 2 L of prime versus 5 L of blood volume in humans). For the conduct of our study, this translated into a reduction of total prime volume from 38 to 10 mL, which significantly decreases the amount of donor blood required to prime the circuit. Although not accomplished in our study, the new rat oxygenator even allows conducting experimental CPB without any donor blood.6 For the current experiments, we chose to use donor blood for priming the circuit because the study was designed to compare the 2 oxygenators with regard to the impact of volume and surface area on outcome alone. However, rats subjected to CPB with the neonate oxygenator obtained more donor blood than rats from the CPB/rat oxygenator group, which could have influenced the perioperative release of inflammatory mediators.37
The modified hole-board test offers the advantage of studying multiple cognitive and behavioral dimensions in a single test and completely avoiding stress.17 In contrast, the Morris water maze is a spatial learning task representing an anxiety-driven test that naturally produces stress, which is a potentially confounding factor.38 Furthermore, the modified hole-board test was successfully used to determine neurocognitive outcome after CPB and deep hypothermic circulatory arrest and after CPB combined with cerebral air emboli in rats.6,39,40 Based on this, and in light of our own data showing that the modified hole-board test is as sensitive as the Morris water maze in detecting cognitive dysfunction after different severities of cerebral ischemia,41 we chose this test environment for the present study.
Even though this model was established to mimic clinical standards as closely as possible, some important limitations remain. To allow the animals' long-term survival, median sternotomy, direct cardiac cannulation, and surgery were not performed. To exclude any effects of temperature on cerebral outcome, CPB was conducted under conditions of normothermia. Young, healthy rats as used for this study naturally lack any comorbidities and significant atheromatous disease as would be found in the aging patient population presenting for cardiac surgery. Although it is difficult to compare the age of rats with the age of humans, undoubtedly, the rats used in this study were comparable to young humans or adolescents, and in these experiments they may be considered comparable to young (adolescent) humans. We decided to study these animals for several reasons: first, we wanted to exclude the effect of atheromatous disease, cardiovascular pathology, or other comorbidities in order to assess the sole effect of CPB on neurocognitive outcome. Second, this age and weight of rats were used to establish the model of CPB in rats, and third, it is this age of rats that is widely used in other studies of cerebral ischemia. Another limitation of this study is that it does not include a complete assessment of the systemic and cerebral inflammatory response to CPB. Instead, with the goal to retain focus, we chose to investigate IL-6 as a systemic inflammatory variable as this is a well-studied variable in cardiac surgery patients. The number of IL-6 measurements was restricted to 3 times to avoid excessive blood loss. With the goal to best characterize the perioperative IL-6 response including its peak, we determined IL-6 at baseline, at the end of CPB (considered the maximum inflammatory response), and 2 h after CPB to assess whether IL-6 release is transient. In addition, NF-κB expression was studied in the hippocampus because this transcription factor plays a key role in regulating the initiation of the inflammatory response and cytokine release in the brain.27,42,43
In summary, this study demonstrates that pronounced systemic inflammation during experimental CPB in young rats is accompanied by an increased expression of NF-κB in hippocampal neurons that is not associated with any neurocognitive impairment. An oversized CPB circuit caused higher levels of systemic IL-6 than an appropriately sized circuit, but this did not result in accentuated cerebral inflammation. These results suggest that factors other than exposure to the extracorporal circulation, such as embolic events, comorbidities, postoperative infection, and age, may play a greater role in contributing to neurocognitive decline. These factors can be simulated and investigated with this animal model to further distinguish between etiologic factors contributing to neurocognitive deficits after CPB and pure epiphenomena.
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