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Anesth Analg 2006;102:1018-1025 © 2006 International Anesthesia Research Society doi: 10.1213/01.ane.0000199221.96250.8c
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Abstract |
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Using a rat model of moderate hypothermic (26°C–28°C) cardiopulmonary bypass (CPB) with hemodilution, we investigated hippocampal apoptotic gene expression and neuronal apoptosis up to 6 h after CPB. The CPB was performed on male rats (380–400 g) under general anesthesia with isoflurane and fentanyl. The right atrium and tail artery were cannulated, and a peristaltic pump and membrane oxygenator were used for CPB. Two groups were studied: Group 1 consisted of fasted rats (n = 15) subjected to 60 min of moderate hypothermic nonpulsatile CPB; Group 2 consisted of sham-operated rats (n = 15). At 1 h after CPB, in 6 rats per group, hippocampus was processed for the apoptotic gene (bcl-2 and bax) messenger RNAs detection by reverse transcriptase polymerase chain reaction, and messenger RNA expression was determined by the ratio of the polymerase chain reaction product of bcl-2 or bax to the ß-actin gene. At 6 h after CPB, in 6 rats per group, hippocampus expression of Bcl-2 and bax protein was determined by immunohistochemistry, and neuronal apoptosis was detected by TUNEL. At 6 h after CPB, in three rats per group, changes in hippocampal CA1 neuronal ultra structure were determined with electron microscopy. Group 1 had increased ratios of bcl-2/ß-actin, bax/ß-actin, and bax/bcl-2 mRNA at 1 h after CPB (bcl-2/ß-actin, 0.82 ± 0.14 versus 0.63 ± 0.07; P = 0.03; bax/ß-actin, 1.04 ± 0.14 versus 0.56 ± 0.03; P = 0.00; bax/bcl-2, 1.31 ± 0.12 versus 0.84 ± 0.09; P = 0.02; Group 1 versus Group 2, respectively). Group 1 had increased bcl-2 and bax protein expression in hippocampal CA1 region at 6 h after CPB (bcl-2, 0.18 ± 0.05 versus 0.09 ± 0.01; P = 0.02; bax, 0.20 ± 0.06 versus 0.04 ± 0.02; P = 0.01; Group 1 versus Group 2, respectively). Group 1 had increased TUNEL staining in hippocampus CA1 at 6 h after CPB (0.14 ± 0.02 versus 0.03 ± 0.01; P = 0.00; Group 1 versus Group 2, respectively). In Group 1 CA1 hippocampus neurons, ultra-structural changes consistent with apoptosis occurred. In rats, moderate hypothermic CPB with hemodilution is associated with CA1 hippocampus bax and bcl-2 gene expression and neuronal apoptosis during the early post-CPB recovery period.
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Introduction |
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Brain injury after cardiopulmonary bypass (CPB) is well recognized (1,2). Purported etiologies for this complication include emboli (3,4), regional hypoxia (5), inflammation (6), and cerebral hyperthermia on rewarming (7,8). Although clinical evidence of cerebral injury has been documented (9,10), the mechanism of injury at the molecular level is unclear.
The hippocampus is an important brain structure involved in recent memory. Hippocampal injury after CPB might explain a portion of the brain injury that has been observed in humans. Neuronal cell death in hippocampus may occur via apoptosis, a process that is likely the consequence of ischemia (11,12). In addition to ischemic etiology, animal studies suggest that neuronal apoptosis may occur after CPB (13). We hypothesized that hypothermic CPB with hemodilution causes hippocampus neuronal injury via apoptosis. The aim of this study was to determine whether hippocampal apoptotic gene expression (bcl-2 and bax) and neuronal apoptosis occurs in rats 1–6 h after hypothermic CPB with hemodilution.
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Methods |
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The Shanghai Second Medicine University (SSMU) Animal Care and Use Committee Approved the experimental protocol. The rat CPB model was adapted from Mackensen et al. (14). Fasted male Sprague-Dawley rats (weight range, 380–400g) were anesthetized with 3% isoflurane in oxygen in a Plexiglas box. After orotracheal intubation, the lungs were mechanically ventilated to maintain a Paco 2 of 35–45 mm Hg. During surgical preparation, anesthesia was maintained with 1.5%–2.0% isoflurane. Rectal temperature was monitored and servo-regulated (by using a variable-temperature water bath-heated blanket) to 37.5°C ± 0.1°C. The right femoral artery was cannulated with a 22-gauge catheter for mean arterial blood pressure monitoring, arterial blood gas analysis, and heparin (500 IU/kg) administration. The tail artery was cannulated with a 20-gauge catheter for use as the arterial inflow during CPB. A multiorifice 14-gauge catheter for venous drainage was inserted in the right internal jugular vein and advanced until the tip of the catheter was placed near the junction of the inferior vena cava and right atrium. The CPB circuit consisted of a venous reservoir, which drained to a peristaltic pump (Mosterflex digital standard drive pump; Cole-Parmer Instrument Company, Vernon Hills, IL). Blood was then pumped through the membrane oxygenator and into the arterial inflow catheter. The same individual performed all surgical preparations (T-JZ).
After surgical preparation, the anesthetic was converted to fentanyl (200 µg/kg) and vecuronium (0.5 mg/kg). A repeat dose of fentanyl (100 µg/kg) was given after 30 min of CPB to ensure adequate anesthesia. After a 15-min stabilization period, the CPB flow was started and maintained at 160–180 mL · kg–1 · min–1, which is similar to the normal cardiac output in the rat. CPB pump flow was continuously measured with an inline flowprobe (2N806 flowprobe and T208 volume flowmeter; Transonics Systems, Inc., Ithaca, NY). Complete CPB was assumed by the absence of pulsatility in the monitoring arterial line. The CPB circuit was primed with approximately 20 mL of bloodless liquid (10 mL of hydroxyethyl starch and 10 mL of Ringer’s lactate solution). The hematocrit during CPB was maintained at 22%–24%. Body temperature was cooled to 28°C within 10 min by a heat exchanger, maintained at 26°C–28°C for 60 min, and then rewarmed to 37°C during a 20-min period with an inflow temperature of 42°C. The outflow temperature was not monitored during rewarming. Only inflow and core temperature were monitored. During CPB, the arterial blood gas values were managed with 
-stat methodology. To prevent atelectasis during CPB, the lungs were not ventilated, but they received 5 mm Hg of continuous positive airway pressure (fraction of inspired oxygen = 0.21). The oxygen tension and temperature used during CPB were chosen to reflect current practice in mainland China during open chamber procedures (15). The rats were subsequently separated from CPB without the need for inotropes or vasopressors. After CPB weaning, the rats received a red blood cell transfusion to maintain the hematocrit at 30%. After decannulation, the rats remained anesthetized with 1.5%–2.0% isoflurane, temperature regulated, intubated, and mechanically ventilated until they were decapitated or perfused by 4% paraformaldehyde, according to the experimental design.
Two groups of rats were studied. Randomization of group assignment was performed using the random digit method. Group 1 consisted of fasted rats (n = 15) subjected to 60 min of moderate hypothermic nonpulsatile CPB. Group 2 consisted of sham-operated rats (n = 15), having undergone identical anesthetic and surgical procedures (including cannulation and heparin administration) except CPB itself, and served as both time and surgical controls. At 1 h after CPB, in 6 rats of each group, hippocampi were removed, homogenized, and processed for apoptotic gene (bcl-2 and bax) messenger (m)RNA detection by reverse transcriptase polymerase chain reaction (PCR), and the expressions of mRNAs were determined by the ratio of the PCR product of bcl-2 or bax to the ß-actin gene. At 6 h after CPB, in 6 rats of each group, hippocampal expression of bcl-2 and bax protein was determined by immunohistochemistry, and neuronal apoptosis was detected by TUNEL staining. At 6 h after CPB, in 3 rats of each group, changes in hippocampal neuronal ultrastructure were determined with electron microscopy. In preliminary experiments, bax and bcl-2 mRNA expression were tested at 30 min, 1 h, and 6 h after CPB. bcl-2 and bax mRNA expression was largest at 1 h. Similar results have been reported in cultured neurons after hypoxic insult (16). Protein expression lags a few hours behind mRNA expression. In rats, bax protein expression was increased 6 h after permanent middle cerebral artery occlusion (17). Given this information, mRNA expression and protein expression were measured at 1 and 6 h after CPB, respectively.
Reverse Transcription-Polymerase Chain Reaction
At 1 h after CPB, rats were maintained deeply anesthetized with 1.5%–2.0% isoflurane before they were decapitated. Another dose of fentanyl (100 µg/kg) was administered IV immediately before decapitation. Sparing the cerebellum and the medulla, the brain was removed, and the left hippocampus was separated rapidly, frozen in liquid nitrogen, and stored at –80°C. Tissue samples were homogenized with 1 mL of Trizol (Invitrogen CO, Carlsbad, CA), and total RNA was extracted. Complimentary (c)DNA was synthesized from 1 µg of pooled mRNA using Oligo-dT Primer (50 pmol), 20 pmol of dNTP, 20 U of RNase Inhibitor (Promega CO, Madison, WI), 200 U of M-MLV RTase (Promega), and 5x buffer. PCR was performed by adding cDNA, 5 pmol of dNTP, bcl-2 or bax primer sense 10 pmol and antisense 10 pmol, ß-actin primer 10 pmol and antisense10 pmol, 16.5 µL of double distilled H2O, and 1 U of Taqase. The subsequent solution was purified by electrophoresis with a 20-gauge/l-agarose gel containing ethidium bromide. The straps of the PCR product were photographed under ultraviolet ray, and the optical density was semiquantitatively analyzed with Tanon GIS gel imaging system (Bio-Tanon CO, Ltd, Shanghai, China). Final numeric values are expressed as a ratio of the gene of interest to the internal (housekeeping) gene, ß-actin.
The following primers for PCR were used: bcl-2 sense, 5'-CTGGTGGACAACATCGCTCTG-3' and antisense, 5'-GGTCTGCTGACCTCACTTGTG-3'; bax sense, 5'-TCCAGGATCGAGCAGA-3' and antisense, 5'-AAGTAGAAGAGGGCAACC-3'; ß-actin sense, 5'-ATTGTAACCAACTGGGACG-3' and antisense, 5'-TTGCCGATAGTGATGACCT-3'. All primers were designed using DNAstar PrimerSelect program (Lasergene, Madison, WI) and synthesized by Shanghai CASarray CO., Ltd (Shanghai, China).
Immunohistochemistry
The deeply anesthetized rats were transcardially perfused using a left ventricular cannula with 100 mL of normal saline solution, followed by 200 mL of freshly prepared 4% phosphate-buffered paraformaldehyde (pH value, 7.4). The left hemi-brains were removed, postfixed in 4% paraformaldehyde for 24 h, and subsequently embedded in paraffin. Only the left hemi-brains were used to minimize possible adverse effects of internal jugular venous cannulation on the right. Sections (4 µm) were harvested from –3.8 mm to –4.16 mm of the bregma for hippocampal bcl-2, bax, and TUNEL staining, respectively. Immunostaining was used simultaneously on a large number of brain sections. Tissue sections (4 µm) were deparaffinized for bcl-2 immunohistochemistry using graded xylene and alcohols, treated with 0.3% H2O2 in methanol (100%) for 25 min, washed in methanol (50%), exposed to sodium borohydride 0.05% in methanol (50%) for 25 min, washed in phosphate-buffered saline (0.1 mol/L, 0.9%, pH 7.4 [PBS]), and microwaved (600 W, to enhance antigen exposure) twice for 5 min in citrate buffer (0.01 mol/L, pH 6.0). After cooling and subsequent washing steps, sections were incubated in 3% normal goat serum in PBS+bovine serum albumin (BSA) at 37°C for 30 min. The tissue was exposed overnight to rabbit polyclonal anti-bcl-2 antibody at a dilution of 1:3,000 in. PBS+BSA at 4°C (bcl-2 [N-19], 200 µg/mL; cat no. sc-492; Santa Cruz Biotechnology, Santa Cruz, CA). On the following day, the sections were first washed twice in PBS+BSA and then incubated with biotinylated goat antirabbit immunoglobulins at a dilution of 1:200 (cat. no. PK 6101; Vector Laboratories, Burlingame, CA) in PBS+BSA for 60 min at 37°C, followed by two washing steps (PBS+BSA and PBS), and then exposed to avidin-biotin-peroxidase (30 min at 37°C). After washing (PBS and distilled water), the tissue sections were visualized with 3,3'-diaminobenzidine (DAB-Chromogen-Kit, cat. no. K 3467; DAKO, Carpinteria, CA), embedded in glycerol gelatin, and coverslips applied. As a negative control, the primary antibody was omitted. The protocol outlined above was also used for bax immunohistochemistry, with the exception of omitting sodium borohydride and using a different dilution (1:800) of the primary anti-bax antibody (rabbit polyclonal immunoglobulin G to bax protein [P-19], 200 µg/mL; cat. no. sc-526; Santa Cruz Biotechnology). The expression of immunohistochemistry was analyzed using the Axioplan 2 imaging system (Zeiss CO, Stuttgart, Germany), and the final value was expressed as the ratio of positive staining area to negative staining area.
TUNEL Staining
TUNEL staining was performed using the In Situ Cell Death Detection Kit, POD (Roche Applied Science, Mannheim, Germany). Paraffin-embedded tissue sections underwent the following protocol: Slides were heated at 60°C followed by xylene wash and a graded series of ethanol and double distilled water washes; protease K incubation for 15–30 min at 21°C–37°C; 1% Triton X-100 washing for 8 min. Slides were rinsed with PBS and incubated for 60 min at 37°C with 50 µL of TUNEL reaction mixture, then incubated for 30 min at 37°C with 50 µL of converter-POD. Slides were rinsed in PBS, then incubated for 10 min at 15°C–25°C with 50 µL of DAB substrate solution, then rinsed again with PBS. After these treatments, the slides were analyzed with light microscopy. The expression of apoptosis was analyzed using the Axioplan 2 imaging system (Zeiss CO), and the final value was expressed as the ratio of positive staining area to negative staining area.
Electron Microscopy
The deeply anesthetized rats were transcardially perfused with 100 mL of normal saline solution, followed by 200 mL of freshly prepared 4% phosphate-buffered paraformaldehyde (pH value, 7.4). The left hippocampus was removed, postfixed in 2.5% glutaraldehyde for 24 h, cut into approximately 1-mm3 pieces, and embedded in resin. Ultramicrotome and electron staining were performed with uranyl acetate and lead citrate prepared ultrathin sections of 50 nm. Ultrathin sections were analyzed under scanning electron microscope for changes in neuronal ultrastructure consistent with apoptosis, including swelling and vacuolization of mitochondria, nuclear pyknosis, and movement of nucleoli to the periphery of the nucleus. A blinded histologist at the Department of Electromicroscopy at the SSMU performed the examinations, and a pathologist in the Department of Pathology at SSMU confirmed the results.
Statistical Analysis
Values were expressed as mean ± sd. Physiologic values were compared between groups using the unpaired Student’s t-test. Differences between groups in the optical density of PCR product of bcl-2/ß-actin and bax/ß-actin, (positive staining area/negative staining area) for protein expressions of bcl-2/bax, and (positive staining area/negative staining area) for TUNEL staining were determined using the Mann-Whitney U-test. A P value of <0.05 was considered significant.
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Results |
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Temperature was lower in Group 1 during CPB. Hematocrit was lower in Group 1 during and after CPB (Table 1).
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Both bcl-2/ß-actin and bax/ß-actin gene expression were increased in Group 1 compared with Group 2 (bcl-2/ß-actin, 0.82 ± 0.14 versus 0.63 ± 0.07; P = 0.03; bax/ß-actin, 1.04 ± 0.14 versus 0.56 ± 0.03; P = 0.00; Group 1 versus Group 2, respectively), and the bax/bcl-2 ratio was larger in Group 1 (1.31 ± 0.12 versus 0.84 ± 0.09; P = 0.02; Group 1 versus Group 2, respectively) (Fig. 1). The electrophoretograms demonstrate increased gene expression of bcl-2 and bax in the CPB group (Fig. 2).
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The ratios of positive area to negative area as the magnitude of bcl-2 and bax protein expression in hippocampus CA1 (Cornu Ammonis, or Ammon’s horn) region were increased in Group 1 (bcl-2, 0.18 ± 0.05 versus 0.09 ± 0.01; P = 0.02; bax, 0.20 ± 0.06 versus 0.04 ± 0.02; P = 0.01; Group 1 versus Group 2, respectively). In hippocampus CA2, CA3, and CA4 regions, bcl-2 and bax protein expression were not different between groups (bcl-2: CA2, 0.05 ± 0.01 versus 0.05 ± 0.01;P = 0.25; CA3, 0.04 ± 0.01 versus 0.03 ± 0.01; P = 0.37; CA4, 0.05 ± 0.02 versus 0.04 ± 0.01; P = 0.32; bax: CA2, 0.03 ± 0.02 versus 0.03 ± 0.01; P = 0.44; CA3, 0.03 ± 0.01 versus 0.03 ± 0.02; P = 0.32; CA4, 0.04 ± 0.02 versus 0.04 ± 0.02; P = 0.13; Group 1 versus Group 2, respectively) (Fig. 3).
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The ratio of positive area to negative area as the magnitude of neuronal apoptosis was increased in hippocampus CA1 of Group 1 (CA1, 0.14 ± 0.02 versus 0.03 ± 0.01; P = 0.001; Group 1 versus Group 2, respectively). In hippocampus CA2, CA3, and CA4 regions, the ratio of positive area to negative area as the magnitude of neuronal apoptosis was not different between groups (CA2, 0.02 ± 0.01 versus 0.02 ± 0.01; P = 0.27; CA3, 0.02 ± 0.01 versus 0.02 ± 0.01; P = 0.09; CA4, 0.03 ± 0.02 versus 0.02 ± 0.01; P = 0.16; Group 1 versus Group 2, respectively) (Fig. 4). Representative slides of hippocampal CA1 TUNEL staining are demonstrated in Figure 5
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Electron microscopy demonstrated obvious abnormalities in the CA1 neuronal ultrastructure of the CPB group, which were consistent with apoptosis. These changes were not present in the control group (Fig. 6). Some mitochondria were moderately to severely swollen with vacuolation and decreased numbers of mitochondrial cristae. Neurons displayed characteristic morphological changes of early apoptosis, including nuclear pyknosis, irregular nucleus, notching of the nuclear membrane, and movement of the nucleoli to the periphery of the nucleus.
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Discussion |
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This study examined markers for neuronal apoptosis in hippocampus after hypothermic CPB with hemodilution in rats. Increases in bax and bcl-2 mRNA were observed one hour after CPB, and increases in bax and bcl-2 protein expression were observed six hours after CPB. Direct evidence of apoptosis in hippocampus CA1 was observed at six hours after CPB by increased TUNEL staining and ultra-structural changes. The results suggest that hypothermic CPB with hemodilution can induce bax and bcl-2 gene expression and neuronal apoptosis in CA1 hippocampus during the early recovery period after CPB. Unlike previous studies that used hypothermic CPB with hemodilution, our study showed, for the first time, regional, as opposed to whole, brain changes in apoptosis-related gene expression.
The strengths of our rat model of CPB are that it may incorporate both hypothermia and hemodilution into a small animal model of CPB. This allows the systematic study of the effect of hypothermia, hemodilution, and CPB on brain injury. The model also allows for long-term recovery. This allows for the determination of histological and behavioral changes. The weaknesses of the model are that inflow occurs at a more distal point in the arterial tree at the tail artery. This compares with inflow occurring in the ascending aorta in CPB with humans. This difference in inflow position may cause changes in brain perfusion. The rat does not develop atheromatous plaque as seen in humans. Cerebral micro-emboli during CPB originating from atheromatous debris will not occur in our rat model. Micro-bubble cerebral micro-emboli could occur in our model, and this issue requires further study.
Our study used a rat model of hypothermic CPB with hemodilution. Previous studies (13) of apoptotic genes in rat cerebral cortex reported that after 90 min of normothermic CPB without hemodilution, expression of apoptosis-related gene mRNAs (bcl-x, bax, caspase 2, and bcl-2) were increased, but no activated caspase 3 protein was detected. Our study differs from this previous study in type of CPB used (hypothermic CPB with hemodilution versus normothermic CPB without hemodilution) and brain region studied (hippocampus versus whole brain). In addition, both neurological and neurocognitive impairment have been reported after 60 minutes of normothermic CPB without hemodilution (14). Whole-blood priming is used in the CPB circuit with normothermic CPB without hemodilution, which may increase systemic inflammation and subsequent brain injury. However, exchange transfusion with CPB priming mixture does not alter systemic interleukin-6 levels or brain cyclooxygenase-2 mRNA expression (18). Hindman et al. (18) described the upregulation of the cerebral cyclooxygenase 2 genes after 60 min of mild hypothermic CPB with hemodilution and found that this change was related to increased systemic interleukin-6 four hours after CPB. Thus, neurological impairment after CPB in rats may be associated with increased whole brain expression of both inflammatory and apoptotic mRNAs.
Our hypothesis was that CPB could cause hippocampus neuronal injury. We demonstrated that CPB induced hippocampus bcl-2 and bax mRNA and protein expression and that there was an imbalance between bcl-2 and bax expression. TUNEL staining and electron microscopy showed evidence of increased CA1 neuronal apoptosis after CPB. All results indicated that CPB could induce apoptosis-related gene expression and resulting neuronal apoptosis in the early recovery period. However, our results should be interpreted with caution. Although it is well documented that the CA1 hippocampus pyramidal neurons are selectively vulnerable to ischemic insults (19), we do not know what changes occurred in other brain regions. The long-term histological changes in hippocampus, if any (14), remain to be defined.
The hippocampus is associated with learning, memory, and cognition, and it is very sensitive to many types of cerebral insults. Neurocognitive dysfunction after CPB impairment has been well documented (1,2). The incidence of cognitive dysfunction immediately after cardiac surgery is reported in the range of 20%–80%, with many patients experiencing long-term or permanent residual deficits (2,20). This prompted us to focus our efforts on the hippocampus. From our study data, we can make no conclusion concerning the relationship between early hippocampal damage and neurocognitive deficits. To test the hypothesis that (at least part of) postoperative cognitive dysfunction is due to hippocampus molecular and ultra-structural changes, it would be important to know whether the current model of hypothermic hemodilutional CPB is accompanied by such postoperative cognitive dysfunction; if it were not, the findings would be of questionable relevance. It would also be important to know whether the current model is associated with comparable changes in other areas of the brain. If that were the case, it would be difficult to postulate a causative relationship between changes in the hippocampus and postoperative neurocognitive dysfunction. Finally, our findings only apply to hypothermic hemodilutional CPB. It remains to be determined how much the findings are caused by CBP, hypothermia, and hemodilution per se.
Apoptosis is the process of programmed cell death. Some genes regulating the pathway of apoptosis, such as bax and bcl-x, promote apoptosis, whereas others (bcl-2 and ced-9) regulate antiapoptotic pathways. It is the balance of these genes in individual neurons that ultimately result in either cell survival or apoptosis (12). We report an increased bax/bcl-2 ratio after CPB, suggesting that apoptosis is favored in hippocampus cells.
There are several plausible reasons that CPB might be expected to induce apoptotic pathways in the brain. Apoptosis contributes to neuronal losses after cerebral ischemia and reperfusion (21). Cerebral ischemia, resulting from multiple cerebral microemboli, occurs in the setting of CPB (3,4). In addition, reactive oxygen species, possibly induced by the systemic and cerebral inflammatory response during CPB (6,18), can directly activate the apoptotic process (22). Other forms of cell stress, such as regional cerebral hypoxia (5) and cerebral hyperthermia during rewarming (8), may occur during hypothermic CPB, which activate apoptotic pathways in the brain. In humans, biochemical evidence of neurological damage has been observed. Serum neuron-specific enolase and S-100 protein, two purported markers of brain injury, have been reported to increase after CPB (9,10). However, the nature of these changes at the cellular and molecular level is not known. Our model of hypothermic CPB with hemodilution is relevant to the clinical condition in humans. The advantage of our study is that we considered the complex effects of CPB, hemodilution, inflammation, and hypothermia on hippocampal injury. It is the interactions of these numerous stresses to the brain that may be responsible for the apoptotic changes we report. The results suggest that the rodent model of moderate hypothermic CPB with hemodilution can induce hippocampal CA1 bax and bcl-2 gene expression and neuronal apoptosis, which may partly explain the mechanism of neurocognitive dysfunction after CPB.
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Footnotes |
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Supported, in part, by Shanghai Municipal Public Health Bureau.
Accepted for publication November 9, 2005.
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References |
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