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Ann Thorac Surg 2006;82:2247-2253
© 2006 The Society of Thoracic Surgeons
a Department of Biochemistry and Biophysics, The University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania
b Department of Pediatrics, The University of Miami, Miami, Florida
c Department of Anesthesia, The Mayo Clinic, Rochester, Minnesota
d Department of Surgery, The University of Oklahoma, Oklahoma City, Oklahoma
e Children’s Hospital of Philadelphia, Department of Anesthesiology and Critical Care, Philadelphia, Pennsylvania
Accepted for publication June 6, 2006.
* Address correspondence to Dr Pastuszko, Department of Biochemistry and Biophysics, 901 Stellar-Chance Bldg, University of Pennsylvania, Philadelphia, PA 19104 (Email: pastuszk{at}mail.med.upenn.edu).
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Abstract |
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METHODS: Animals were assigned to sham operation and three experimental groups. The experimental groups were placed on bypass, cooled to 18°C, and subjected to 90 minutes of deep hypothermic circulatory arrest (DHCA), low-flow cardiopulmonary bypass (LFCPB) at mL/(kg · min), or selective cerebral perfusion (SCP) at 20 mL/(kg · min), followed by rewarming and 2 hours of recovery. The oxygen pressure in the microcirculation of the cortex was measured by quenching of phosphorescence. Levels of phosphorylated and total protein were determined by Western blot analysis.
RESULTS: Control oxygen pressure was 55 ± 9 mm Hg and decreased during DHCA, LFCPB, and SCP to 1.1 ± 0.6 mm Hg, 9.8 ± 2.3 mm Hg, and 9.3 ± 1.9 mm Hg, respectively (p < 0.001). After DHCA, N-terminal of Bcl-2-associated X protein (N-Bax) levels increased (295% ± 15%, p < 0.01), B-cell leukemia protein (Bcl-2) levels decreased (31% ± 9%, p < 0.01), and phosphorylation level of protein kinase B (pAkt) and extracellular signal-regulated kinase 1/2 (pERK1/2) did not change. After LFCPB and SCP, N-Bax and Bcl-2 levels were unchanged, pAkt levels increased (367% ± 122%, p < 0.05 and 337% ± 47%, p < 0.01, respectively), pERK1 (484% ± 70% and 501% ± 255%, respectively; p < 0.01) and pERK2 (569% ± 128%; p < 0.001 and 494% ± 162%; p < 0.05, respectively) levels increased, and total ERK2 levels also increased (279% ± 90% and 153% ± 44%, respectively, p < 0.05).
CONCLUSIONS: Stable levels of Bcl-2 and Bax and the increases in pAkt and pERK1/2 after LFCPB and SCP are likely indicators of improved chances for cell survival.
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Introduction |
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Several transcription proteins critical in the regulation of survival pathways or in cell death after hypoxia/ischemia have been identified, including the B-cell leukemia protein family (Bcl-2), protein kinase B (Akt), and mitogen-activated protein kinase family (MAPK). The Bcl-2 family includes both proapoptotic and antiapoptotic proteins [1]. The balance between these two proteins of the Bcl-2 family largely determines the survival or death of a cell. The Akt is a component of many receptor signal-transduction pathways and can prevent cell death after growth factor withdrawal [2]. Akt exerts its antiapoptotic effects through several downstream targets, including the cyclic adenosine monophosphate response element-binding protein (CREB). The MAPK family plays crucial roles in signal transduction from the cell surface to the nucleus and regulates cell death and survival. One member of the MAPK family, the extracellular signal-regulated kinase 1/2 (ERK1/2), has been proposed to play an important role in the pathogenesis of cerebral ischemic injury [3, 4] and also in neuroprotection [5]. The mechanisms of ERK1/2-mediated neuroprotection involve activation of the antiapoptotic proteins and inhibition of apoptotic proteins.
The goal of the present study was to determine expression of Bcl-2, an antiapoptotic protein, and Bax, a proapoptotic protein, and also changes in phosphorylation of Akt and ERK1/2 in the striatum of newborn piglets after DHCA, LFCPB, and SCP. We hypothesized that a prolonged period of severe hypoxia (DHCA) compared with mild hypoxia (LFCPB and SCP) would promote apoptosis and neuronal injury in the newborn brain.
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Material and Methods |
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All animal procedures were in strict accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, 1996) and were approved by the local Animal Care Committee.
Cardiopulmonary Bypass Technique and Experimental Protocol
Full CPB flow was set at 125 mL/(kg · min). Alpha-stat strategy was used in all CPB piglets to mimic the clinical setting used in the operating room. Anesthesia was maintained during CPB with isoflurane at 0.5 to 1.0 volume percentage, repeated intravenous (IV) pancuronium boluses of 0.1 mg/kg, and an IV fentanyl infusion of 10 µg /(kg · h). The mean blood pressure on full support was typically 50 to 60 mm Hg, and the hemoglobin was maintained at 10 to 11 g/dL. During recovery, mean blood pressures were maintained at the prebypass level of 70 to 80 mm Hg with normal saline and 5% albumin. No inotropic agents were used.
Once CPB was begun, the animals were cooled to a nasopharyngeal temperature of 18°C over 30 minutes. Ventilation was stopped after initiation of CPB.
Low-Flow hypothermic cardiopulmonary bypass
When the temperature reached 18°C, the CPB circuit flow was reduced to 20 mL/(kg · min). After 90 minutes of LFCPB, the flow was gradually increased again to 125 ml/(kg · min), and the piglets were rewarmed to 36°C over a 30-minute period.
Deep hypothermic cardiac arrest
When the temperature reached 18°C, CPB was stopped. After 90 minutes of DHCA, the flow was gradually increased again to 125 ml/(kg · min), and the piglets were rewarmed to 36°C over a 30-minute period.
Selective cerebral perfusion
Before the piglets were placed on CPB, the aortic arch and its vessels were exposed. Once the piglets reached the appropriate study temperature, the ascending aorta, transverse aorta, and left carotid artery were cross-clamped. The arterial cannula was advanced into the right carotid artery and the bypass flow rate was lowered to 20 ml/(kg · min). After 90-minute period, the cross-clamps were removed, the cannula was moved back into the ascending aorta, and CPB was slowly increased to 125 ml/(kg · min) during the 30-minute rewarming period.
Measurements of Oxygen Pressure and Oxygen Distribution by the Oxygen-Dependent Quenching of Phosphorescence
The cortical oxygen pressure was measured by oxygen-dependent quenching of phosphorescence [6, 7]. Oxyphor G2 (6 mg/mL in physiologic saline, pH 7.4; Oxygen Enterprises, Ltd, Philadelphia, PA) was intravenously injected (4.0 mg/kg body weigh). The phosphorescence lifetime measurements were made using a frequency domain PMOD 5000 phosphorometer (Oxygen Enterprises, Ltd.) with a 635 nm light-emitting diode as an excitation source and an avalanche photodiode (APD) detector. The phosphorescence signal was passed through a 695 nm long-pass filter (3 mm Schott glass, Schott North America, Elmsford, NY). The excitation light was conducted to the tissue and the phosphorescence collected by 3-mm-diameter light guides. The tips of the light guides were placed very near the surface of the dura with an 8-mm center-to-center separation. The measurements are of the mean oxygen pressure in the microvasculature.
Western Blot Analysis
Samples of frozen striatal tissue were homogenized in a buffer containing 2% sodium dodecyl sulfate (SDS), 10 mM Tris-HCl (pH 7.4) freshly supplemented with sodium fluoride (10 mM), sodium pyrophosphate (10 mM), sodium orthovanadate (1 mM), sodium molybdate (1 mM), phenylarsine oxide (1 µM), and aprotinin (10 µg/mL). The homogenate was boiled for 5 minutes after addition of SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer.
Protein concentration was determined in a homogenate aliquot with a BCA Protein Assay kit (Pierce, IL). An equal amount of protein for each sample was separated by 12% SDS-PAGE and transferred onto a nitrocellulose membrane (Hybond C, Amersham Pharmacia Biotech, Piscataway, NJ). The membranes were then incubated in a blocking solution of phosphate buffered saline (PBS), pH 7.4, containing 5% nonfat milk powder for 1 hour at room temperature. The membranes were then incubated overnight with specific antibodies: ERK1 (cross-reacting with ERK2), Bax (N-20), Bcl-2 (Santa Cruz Biotechnology, Santa Cruz, CA); phospho-ERK1/2, phospho Akt, Akt (Cell Signaling Technology, Beverly, MA), and ß-actin (ABCAM, Cambridge, MA), which served as a loading control. After being washed in PBS containing 0.05% Tween 20 (PBS-T; Sigma-Aldrich, St. Louis, MO), the membranes were incubated for 1 hour with peroxidase-conjugated goat antirabbit or antimouse immunoglobulin G (Amersham Pharmacia Biotech). The final reaction was visualized using enhanced chemiluminescence (ECL Western Blotting Detection Reagents, Amersham Pharmacia Biotech.), and the membranes were exposed to x-ray film.
Data Analysis
Autoradiographic films were analyzed using Scion Image software (NIH, Bethesda, MD). Each blot contained two sets of samples, one for an experimental group and another for the control group. The data were normalized to the values obtained for the untreated control group (assigned a value of 100). Statistical analysis was performed using one-way analysis of variance, followed by the Mann-Whitney test. Statistical significance was set at p < 0.05.
All physiologic parameters and cortical oxygen pressure values are expressed as means for "n" experiments ± SD. Statistical significance was determined using one-way analysis of variance with repeated measures by Wilcoxon signed-rank test, and p < 0.05 was considered statistically significant.
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Results |
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Low-Flow hypothermic cardiopulmonary bypass
During LFCPB, the pH increased to 7.59 ± 0.08 (p < 0.05), PaCO
2 decreased to 18.3 ± 4.8 mm Hg (p < 0.05), and PaO
2 increased to 197.0 ± 23.6 mm Hg (p < 0.05). All blood gas values returned to prebypass levels during the recovery period. The MABP decreased to 20 ± 5 mm Hg (p < 0.005) and then, during rewarming and postbypass recovery, steadily increased. By the end of the experiments, the MABP was 63.4 ± 8 mm Hg (p < 0.05), significantly below control value.
Selective cerebral perfusion
During SCP, the pH increased to 7.79 ± 0.04 (p < 0.05), but rewarming and postbypass recovery values were not significantly different from control. PaCO
2 decreased significantly during SCP to 14.1 ± 2.2 mm Hg (p < 0.001), but PaO
2 significantly increased during SCP to 169.3 ± 18.6 mm Hg (p < 0.05). Both parameters returned to control values during recovery. The MABP decreased to 16.5 ± 4.2 mm Hg (p < 0.001) at the end of SCP and returned to control values during rewarming.
The prebypass cortical oxygen pressure was 55 ± 9 mm Hg. The cortical oxygen pressures during DHCA, LFCPB, and SCP are shown in Figure 1. The values are reported as the peak of the histograms, since this most accurately represents the microcirculation within the cortical tissue. In each case, the values were corrected to the appropriate tissue temperature (18°C for DHCA, LFCPB, and SCP). The oxygen pressure decreased during the first 5 minutes of DHCA to 2.9 ± 1.7 mm Hg (p < 0.001 versus baseline control) and was 1.1 ± 0.6 mm Hg (p < 0.001) at the end of arrest.
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Effects on Cell Regulators in Striatum
Levels of Bcl-2
As shown in Figure 2, Bcl-2 immunoreactivity was decreased after DHCA (31% ± 9% compared with control, p < 0.01) and did not show significant changes after LFCPB and SCP (79% ± 21% and 94% ± 25%, respectively).
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Levels of Akt
After DHCA, the phospho-Akt (pAkt) immunoreactivity did not change (121% ± 67% compared with control), as can be seen in Figure 3. LFCPB induced a significant increase in pAkt immunoreactivity (367% ± 122% compared with control (p < 0.05). SCP also caused an increase in pAkt immunoreactivity (337% ± 47%, p < 0.01; Fig 3). Measurements of total Akt immunoreactivity did not show any differences between the control and the experimental groups (Fig 3).
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To determine the extent to which the ratios of phosphorylated to dephosphorylated ERK1/2 were altered, phospho-ERK1/2 levels were normalized to the total ERK1/2. Normalized pERK1 and pERK2 levels were significantly increased in the SCP group (308% ± 30% and 467% ± 51%, respectively, p < 0.01), and normalized pERK2 levels were significantly increased in the LFCPB group (466 ± 14, p < 0.01) compared with those in control group.
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Comment |
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Bax expression is altered by cerebral ischemia [8, 9], suggesting a role in ischemic neuronal death. Translocation of Bax from the cytosol to mitochondria is a central event of the apoptotic process. How exactly Bax initiates apoptosis is unknown, although the opening of channels in the mitochondrial membrane with release of peptogenic factors is probably important [10]. Using an antibody (N-20) specific to the conformationally changed (proapoptotic) state, we demonstrated that proapoptotic Bax expression was elevated after DHCA but unaltered after LFCPB or SCP. Our data clearly show early proapoptotic events after an ischemic episode. These data suggest that ischemic conditions during and after 90 minutes DHCA were severe enough to increase the risk of apoptosis within the striatum, whereas the perfusion of the brain provided by LFCPB and SCP spared activation of apoptosis.
The increase in pAkt levels associated with LFCPB and SCP may be important for neuroprotection after these procedures. Akt activation appears to protect neurons from damage after ischemia [11] and is required for neuronal survival mediated by ischemic preconditioning [12, 13]. Akt inhibits Bax conformational change and its redistribution to the mitochondrial membranes. The lack of Akt activation after DHCA could therefore contribute to the increase in proapoptotic Bax, whereas increased pAkt after LFCPB and SCP aids in maintaining the level of proapoptotic Bax within normal range.
The observed increases in ERK1/2 phosphorylation after LFCPB and SCP and the trend toward elevated phosphorylation after DHCA could have major significance on the recovery associated with these procedures. ERK1/2 is an important intermediate in cellular regulation and is thought to have roles in neuronal death and survival, although these roles seem to be complicated and altered by the type of cells and the magnitude and timing of insults [14, 15]. The activation of ERK has been reported to protect neurons against transient forebrain ischemia [16]. Furthermore, pERK1/2 is also involved in antiapoptotic activity [17] and in the development of ischemic tolerance [18].
On the other hand, several lines of evidence suggest that sustained ERK activation may indicate serious neuronal injury, possibly leading to cell death [19]. Inhibition of the ERK pathway has been shown to decrease cerebral ischemic–reperfusion injury [20]. Aharon and colleagues [4] connected the increase in expression of pERK1/2 in cerebral vascular endothelium with neuronal damage in the brain of piglets after 24 hours of recovery from normothermic CPB, DHCA, and LFCPB and concluded that ERK1/2 may play a prominent role in early cerebral ischemic–reperfusion injury. We suggest, however, that the increase in pERK induced by LFCPB and SCP can play a role in neuroprotection, whereas the lack of significant increase in pERK phosphorylation after DHCA is related to the development of striatal injury. This latter speculation is based on our previously published results that indicate at 2 hours of recovery, LFCPB [21] and SCP [22], but not DHCA [21], cause a significant increase in CREB phosphorylation and expression.
Previous reports suggest that prolonged ERK activation accompanied by an increase in CREB can be linked to cell survival and plasticity [23–25]. ERK1/2 is an upstream kinase of transcription factor CREB. CREB activation through phosphorylation induces expression of proteins involved in protective mechanisms and is associated with cell survival. Although CREB phosphorylation can be induced by other kinases, activation of ERK1/2 is necessary for inducing expression of CREB target genes, which include the anti-apoptotic protein Bcl-2. Thus, it appears that the relationship of ERK and CREB phosphorylation may ultimately determine whether cells die or survive within a given region.
The observed increases in Akt and ERK1/2 phosphorylation after LFCPB and SCP, combined with an increase in CREB phosphorylation and stable levels of Bax and Bcl-2, could be related to the tissue oxygenation. Our previous studies and results in the present study show that the peak of the brain tissue oxygen histograms was near zero during DHCA, whereas the peaks were at about 9 mm Hg during LFCPB and SCP. Thus, after 90 minutes of DHCA in which brain tissue was severely ischemic, there were no significant increases in pAkt, pERK1/2, and CREB. The latter was also accompanied by an increase in activated Bax and decreased in Bcl-2. In contrast, in LFCPB and SCP, the pAkt, pERK1/2, and CREB increased, and Bax and Bcl-2 levels remain unchanged where the hypoxia was much milder.
A limitation of this study is that we investigated only the first steps of the intrinsic (mitochondrial) signal pathway leading to apoptosis. Future experiments with a longer recovery (up to few days) will allow us to characterize the activity of caspases and the cascade of proteolytic activity after the experimental conditions we created and also validate our results with histopathologic data.
In conclusion, we suggest that the combined increases in Akt, ERK1/2, and CREB phosphorylation in the striatum after LFCPB and SCP is a likely indicator of improved chances for cell survival compared with the increase in proapoptotic form of Bax and decrease in Bcl-2 after DHCA. As such, these variables are indicative of and provide further insight into the mechanisms underlying the response of the striatum to ischemic stress caused by heart surgery. We also speculate after prolonged DHCA (90 minutes), that severe hypoxia alters gene expression and protein phosphorylation promoting apoptosis in the brain and cell death.
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Acknowledgments |
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