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Anesth Analg 2002;94:1389-1394
© 2002 International Anesthesia Research Society

CARDIOVASCULAR ANESTHESIA

Differential Cerebral Gene Expression During Cardiopulmonary Bypass in the Rat: Evidence for Apoptosis?

Yukie Sato, MD*, Daniel T. Laskowitz, MD{dagger}, Ellen R. Bennett, PhD{ddagger}, Mark F. Newman, MD*, David S. Warner, MD*, and Hilary P. Grocott, MD FRCPC*

Departments of *Anesthesiology, {dagger}Medicine (Neurology), and {ddagger}Pathology, Duke University Medical Center, Durham, North Carolina

Address correspondence and reprint requests to Hilary Grocott, MD, Associate Professor of Anesthesiology, Department of Anesthesiology, Duke University Medical Center, Box 3094, Durham, NC 27710. Address e-mail to h.grocott{at}duke.edu


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Cardiopulmonary bypass (CPB) is associated with a spectrum of cerebral injuries. The molecular changes in the brain that might contribute to these injuries are not clearly known. We sought to determine whether the expression of apoptotic genes is increased after CPB in the rat. Rats (n = 7) were subjected to 90 min of normothermic CPB. A group of sham-operated rats (n = 7) served as non-CPB controls. After a 3-h post-CPB period of recovery, their brains were removed, homogenized, and processed for messenger RNA (mRNA) extraction. By using a ribonuclease protection assay, the ratios of both pro- and antiapoptotic mRNA (bcl-x, bcl-2, bax, caspase 2, and caspase 3) to the housekeeping glyceraldehyde phosphate dehydrogenase (GAPDH) gene were determined. Additionally, Western immunoblotting was performed to detect the presence of activated caspase 3, a protein central in the apoptotic process. Compared with the non-CPB controls, the CPB group had significantly increased levels of apoptotic/GAPDH mRNA ratios (bcl-x, 0.414 ± 0.152 CPB versus 0.251 ± 0.051 non-CPB, P = 0.048; caspase 2, 0.030 ± 0.014 CPB versus 0.018 ± 0.005 non-CPB, P = 0.048; bax, 0.106 ± 0.035 CPB versus 0.066 ± 0.009 non-CPB, P = 0.009; bcl-2, 0.011 ± 0.006 CPB versus 0.006 ± 0.002 non-CPB, P = 0.035). However, no activated caspase 3 protein was detected in either group. Elucidating the molecular biological sequelae of CPB may aid in the understanding of the pathophysiology of cardiac surgery-associated cerebral injury and, in doing so, may be useful in identifying potential therapeutic targets for pharmacologic neuroprotection.

IMPLICATIONS: Cardiopulmonary bypass (CPB) appears to induce transcription of pro- and antiapoptotic genes in the rat brain, but caspase-mediated apoptosis itself does not appear to be activated. Elucidating the molecular biological sequelae of CPB may aid in the understanding of the pathophysiology of cardiac surgery-associated cerebral injury and, in doing so, may be useful in identifying potential therapeutic targets for pharmacologic neuroprotection.


   Introduction
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 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Adverse cerebral outcomes after cardiopulmonary bypass (CPB) for cardiac surgery have been well documented (1–7). These injuries encompass a complete spectrum, from subtle cognitive impairment to overt stroke. The etiology of these cerebral injuries, although not clearly understood, probably represents a complex interaction among cerebral microemboli (8,9), global cerebral hypoperfusion (10), inflammation (11), cerebral temperature modulation (12–15), and genetic susceptibility (16). Although the cerebral consequences of CPB have been measured clinically, insights into the molecular events within the brain occurring as a result of CPB have only begun to be investigated (17,18).

Apoptosis is a well documented series of events that results in the programmed self-destruction of cells. Its stimulus for initiation is variable but includes ischemia and other stresses (19,20). These stresses induce an intracellular molecular cascade that ultimately results in self-destruction of tissue. Apoptosis is responsible for the long-term loss of neuronal tissue after cerebral ischemia (21), but its potential role in CPB-associated cerebral injury is not clearly known.

Gaining insights into the cerebral consequences of CPB, and in particular the molecular pathways possibly involved, has been limited by the relative inability to study brain tissue after recovery from CPB. With the development of a rat model of CPB (22,23), we have been able to sample brain tissue after CPB for molecular analysis with well established molecular tools. In this study, we hypothesized that the transcription of apoptotic genes would be increased after CPB and that protein evidence for apoptosis would confirm the initiation of this self-destructive cellular process.


   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
After Duke University Animal Care and Use Committee approval, the following experiment (adhering to the Guide for the Care and Use of Laboratory Animals (24) was performed.

With a CPB model that we developed and described previously (22,23), fasted male Sprague-Dawley rats (325–375 g; Harlan, Indianapolis, IN) were anesthetized with 3% isoflurane in a Plexiglas® box. The trachea was subsequently intubated and the lungs mechanically ventilated to maintain a PaCO2 of 36–42 mm Hg. The arterial blood gas values were managed with {alpha}-stat methodology. During surgical preparation, anesthesia was maintained with 1.5%–2.0% isoflurane.

Rectal temperature was monitored and servoregulated (by using a forced-air convective system and variable-temperature water bath-heated blanket) to 37.5°C ± 0.1°C. The left carotid artery was cannulated with PE-50 tubing and used for mean arterial blood pressure monitoring and arterial blood gas analysis. Occlusion of a unilateral carotid artery has not been shown to affect ipsilateral CBF in rats (25) or histologic outcome after CPB in rats (26), presumably because of an intact circle of Willis. Heparin (150 IU IV) was given after placement of the first intravascular catheter. The tail artery was cannulated with a 20-gauge IV catheter to serve as the arterial inflow for CPB. Through a horizontal neck incision, the right internal jugular vein was cannulated with a 4.5F dual-stage venous cannula. This multiorificed venous cannula was inserted and advanced until the cannula tip was placed at the junction of the inferior vena cava and right atrium (the position of which was confirmed in pilot experiments by using transesophageal echocardiography) (22). This allowed for excellent venous drainage of the inferior vena cava, superior vena cava, and coronary sinus (which drains the persistent left superior vena cava in rats).

The CPB circuit consisted of a venous reservoir that was drained to a peristaltic pump (Masterflex®; Cole-Parmer Instrument Co., Vernon Hills, IL) that pumped the blood to a membrane oxygenator (a modified Cobe Micro® oxygenator; Cobe Cardiovascular, Inc., Arvada, CO) and then to the arterial inflow cannula. CPB pump flow was continuously measured with an in-line flowprobe (2N806 flowprobe and T208 volume flowmeter; Transonics Systems, Inc., Ithaca, NY).

No blood incompatibilities are known between Sprague-Dawley rats, thereby allowing the CPB circuit to be primed with 40 mL of whole blood from two heparinized donor rats phlebotomized under isoflurane anesthesia. If needed, up to 3 mL of 6% hetastarch (HextendTM; Abbott Laboratories, North Chicago, IL) was added to the circuit to maintain a blood level of 2–3 mL in the venous reservoir. Mixed venous oxygen saturation (from the venous return line in the CPB group only) was measured continuously with an Oximetrix® monitor (Abbott Laboratories) and an in vivo-calibrated 4F Opicath® catheter (Abbott Laboratories).

After surgical preparation, the anesthetic was converted to fentanyl (150 µg/kg IV), diazepam (2 mg/kg IV), and pancuronium (0.2 mg IV). A repeat dose of fentanyl (75 µg/kg IV) and diazepam (1 mg/kg IV) was given after 30 min of CPB to ensure adequate anesthesia. This anesthetic has been shown in pilot experiments (supervised by our institutional veterinarians) to prevent physical movement (escape behavior) to painful stimuli in the unparalyzed rat.

After 15 min of stabilization, CPB was performed for 90 min, and the rats were subsequently separated from CPB without the need for inotropes or vasopressors. After decannulation, the rats remained anesthetized, temperature regulated, intubated, and mechanically ventilated for 3 h, after which they were decapitated.

Two groups of animals were studied. The CPB group (n = 7) underwent all of the previously described procedures. A group of sham-operated animals (n = 7), having undergone identical anesthetic and surgical procedures (including cannulation) except CPB itself, served as controls.

For messenger RNA (mRNA) extraction and analysis, after decapitation, the brain was excised; the right cerebral cortex was removed and divided into two equal representative samples. One sample was homogenized with 5 mL of Trizol (Gibco BRL, Rockville, MD) per the commercial RNA extraction kit-recommended protocol, with the other sample reserved for Western immunoblotting. The samples were frozen at -80°C until later analysis.

All of the samples were analyzed with a commercially available multiprobe ribonuclease protection assay kit (RiboquantTM; PharMingen, San Diego, CA). Briefly, an antisense RNA probe specific for the target mRNA was constructed, and the RNA extract was added and mixed, allowing for hybridization. The binding of the sense and antisense RNA offered protection from the ribonucleases that were added to the solution. The subsequent solution was purified and, with polyacrylamide gel electrophoresis, the individual mRNA targets were separated and identified. Digitized data were collected and analyzed with the Molecular Dynamics (Sunnyvale, CA) Storm Phosphor Image System and the ImageQuantTM software analysis package. The image density of the corresponding mRNA fragments allowed for quantification of the mRNA. The apoptotic genes identified were bcl-2, bcl-x, bax, caspase 2, and caspase 3. To control for loading conditions, final numeric values are expressed as a ratio of the gene of interest to the internal (housekeeping) gene, glyceraldehyde phosphate dehydrogenase (GAPDH).

The second brain sample was analyzed concurrently by using Western immunoblotting to detect activated caspase 3 protein. Rat brain homogenates were generated by Dounce homogenization of tissue in ice-cold homogenization buffer. The protein concentration was determined with the Bio-Rad Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA). Immunodetection of the large fragment of activated caspase 3 in a standard 20-µg aliquot of protein homogenate (subsequently increased to 30 µg to ensure an optimal protein signal) was performed by using cleaved caspase 3 (D175) antibody (Cell Signaling Technology, Beverly, MA) by following the manufacturer’s recommendation. A fluorescein-linked antispecies secondary antibody followed by an antifluorescein alkaline phosphatase conjugate and ECF substrate was used for protein detection according to the ECF Western Blotting Kit (Amersham Life Science, Piscataway, NJ). The dry membranes were scanned on a Storm 860 Phosphor Imager by using a 570-nm filter and ImageQuant 5.1 software.

Physiologic values were compared between groups by using a repeated-measures analysis of variance. When significant, between-group comparisons were made by using a Student’s t-test. Differences in the optical density of apoptotic mRNA and protein images were compared by using the Mann-Whitney U-test. A P value of <=0.05 was considered significant.


   Results
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 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Physiologic values are represented in Table 1. There were no statistically significant differences between groups, with the exception of temperature. During CPB, the CPB group temperature was slightly lower, albeit within a normothermic range, than that of the non-CPB group (37.1°C ± 0.4°C CPB versus 37.5°C ± 0.1°C non-CPB; P = 0.01).


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  		Table 1. Physiologic Values
 
Gene expression is summarized in Figure 1, which represents the mRNA ratios of the five apoptotic genes examined. Messenger RNA encoded by four of the five apoptotic genes examined was increased in the CPB group after 3 h of CPB. The bax, bcl-2, caspase 2, and bcl-x to GAPDH ratios were significantly increased compared with controls (bax, 0.106 ± 0.035 CPB versus 0.066 ± 0.009 non-CPB, P = 0.009; bcl-2, 0.011 ± 0.006 CPB versus 0.006 ± 0.002 non-CPB, P = 0.035; caspase 2, 0.030 ± 0.014 CPB versus 0.018 ± 0.005 non-CPB, P = 0.048; bcl-x, 0.414 ± 0.152 CPB versus 0.251 ± 0.051 non-CPB, P = 0.048). There was a trend toward caspase 3 being increased as well, but it did not achieve statistical significance (0.042 ± 0.018 CPB versus 0.026 ± 0.004 non-CPB; P = 0.11). Western immunoblotting failed to demonstrate any detectable activated caspase 3 in either the CPB or non-CPB control groups (Fig. 2).



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  		Figure 1. Apoptotic messenger RNA 3 h after 90 min of cardiopulmonary bypass (CPB); bcl-x, caspase 2, bax, and bcl-2 were significantly increased (P = 0.048, 0.048, 0.009, and 0.035, respectively). Caspase 3 differences did not achieve statistical significance (P = 0.11). GAPDH = glyceraldehyde phosphate dehydrogenase.

 


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  		Figure 2. A representative Western immunoblot analysis of rat brain homogenates 3 h after 90 min of cardiopulmonary bypass (CPB) with cleaved caspase 3 antibody for detection of activated caspase 3. A, Non-CPB group; B, CPB group. Lane 1= negative control; Lane 2 = positive control; Lanes 3–9 = individual animals from each experimental group. The arrow denotes the presence of activated caspase 3 in the positive lane. The Western immunoblotting did not show any for the presence of activated caspase 3 protein in either the CPB or Non-CPB groups.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Functional cerebral injury after CPB is a well recognized clinical entity (3,5,6). Cognitive impairment has been demonstrated in 80% or more of patients immediately after cardiac surgery but, more importantly, has been present in up to 40% of patients months after surgery, with many patients experiencing permanent residual deficits (7). This suggests that long-term changes in the brain may be initiated as a result of CPB. The nature of these changes at the cellular and molecular level, if these are indeed present, is not known. However, the initiation of an apoptotic process that results in delayed loss of neurons may be a possible explanation for the occurrence of these long-term injuries and prompted this investigation. We have demonstrated that CPB in rats induces increases in the expression of several genes responsible for the control of apoptosis. However, whether apoptosis actually occurs is not clear. We could not demonstrate the presence of activated caspase 3, suggesting that if apoptosis ultimately does occur as a result of the genes with increased expression, it does not appear to be caspase mediated.

The molecular events initiated as a result of CPB are only beginning to be described. Bokesh et al. (17) first described the transcription of several immediate response genes resulting from a combination of CPB and global cerebral ischemia induced during hypothermic circulatory arrest in lambs. In their study, they described upregulation of c-fos and c-jun mRNA, whose protein products function as transcription regulators of genes affecting cell survival and death (27). However, they did not completely describe whether these events resulted from the CPB alone, hypothermic circulatory arrest alone, or a combination of both. Hindman et al. (18) have described the upregulation of one of the inflammatory genes in the brain during CPB. Using a similar CPB and mRNA analysis technique as outlined herein, they described the cerebral expression of inducible cyclooxygenase 2 after CPB.

We have developed a survival model of CPB in the rat that allows for the investigation of molecular biological changes in the brain (22,23). In this model, we have observed neurologic and neurobehavioral deficits, comparable to postcardiac surgery cognitive deficits seen clinically, after CPB in rats. These deficits have been demonstrated after as little as 45–60 minutes of normothermic CPB in rats. One of the advantages of using the rat is that it allows the use of the vast array of previously described molecular techniques to dissect, at the molecular level, the cerebral consequences of CPB.

Apoptosis is a well documented series of events that results in the programmed self-destruction of cells (20). There are several plausible reasons that CPB might be expected to induce apoptotic pathways in the brain. In the setting of reperfusion after cerebral ischemia, apoptosis contributes to neuronal losses (21). Cerebral ischemia, resulting from multiple cerebral microemboli, is clearly possible in the setting of CPB. In addition, reactive oxygen species, possibly induced by the systemic and cerebral inflammatory response seen during CPB (11,18), can directly activate the apoptotic process (28).

The transcription of apoptotic genes into their respective mRNA is only a preliminary step in the complete process of apoptosis (29,30). These mRNAs must be translated into protein, which may or may not require posttranslation modification before ultimately acting to complete apoptotic pathways. We demonstrated that several mRNAs are present after CPB. Some (bax, caspase-2, caspase-3) encode for proapoptotic pathways, whereas others (bcl-2, bcl-x) encode for antiapoptotic pathways. It is the balance of these genes in individual neurons that ultimately results in either cell survival or death. It is important, therefore, to look more definitively at a step further down the apoptotic pathway than just the expression of these genes. A near to final step in this process is the cleavage of caspase 3 into its active form (31–33); without this, caspase-dependent apoptosis cannot occur (34). We therefore used Western immunoblotting to look for this protein in brain tissue homogenates. Its absence highly suggests that although CPB induced multiple genes to be differentially expressed, the apoptotic pathway was not ultimately completed. It is possible that the respective effects of the pro- and antiapoptotic genes were balanced, thus accounting for the lack of cleavage of caspase 3 into its activated form.

There were several limitations in this study. Although apoptosis has generally been considered to involve caspase 3-dependent pathways (20), evidence is now emerging that caspase-independent pathways may also lead to apoptosis (35). It is possible that apoptosis does occur after CPB, but if so, it is not caspase 3 related. We did not investigate this newly discovered apoptotic pathway.

Another limitation relates to the small physiological differences between groups. There was a small temperature difference between the groups, with the CPB group being, at times, 0.4°C cooler than the Non-CPB group. However, it is unlikely that this small temperature difference had any effect, particularly because even with this lower temperature, the temperature ranges during CPB were within an accepted normothermic range. One other limitation relates to the time window during which we sampled brain for gene changes. The temporal pattern of these gene expression changes is unknown. This apoptotic expression may be a transient surge and not result in any long-term changes. The three-hour time period during which we looked for the presence of apoptotic changes may have been too early to see effective changes. Previous CPB models, albeit circulatory arrest models, have identified the occurrence of apoptosis, but at a time period six hours after CPB (36).

The spatial pattern of this differential gene expression is also unknown. To avoid any potentially confounding factors caused by cannulation of the left carotid artery (25), we examined the entire right cerebral cortex as a whole. It may be that various areas of brain differentially produce this mRNA, resulting in a variety of functional effects, one of which potentially is neurocognition. If the hippocampus is responsible for the mRNA signal, a link between neurocognitive decline mechanisms might be obtained. However, if other functionally silent areas of the brain are primarily responsible for the increased mRNA signal, then a neurocognitive correlate is less likely.

With the demonstration in this study (as well as others) that gene expression changes are initiated as a result of CPB, insights into the mechanism of cerebral injury during cardiac surgery may be found. Understanding how these pathways are initiated and the effects they produce may lead to the development of pharmacologic neuroprotectant drugs.


   Acknowledgments
 
Dr. Grocott was supported by a Pepper Center Junior Faculty Award (NIA-AG11268).


   References
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 Introduction
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 Results
 Discussion
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Accepted for publication February 13, 2002.




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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins and Stanford University Libraries' HighWire Press®. Copyright 2002 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press