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Ann Thorac Surg 2004;78:1418-1425
© 2004 The Society of Thoracic Surgeons

Original article: cardiovascular

Increased Transcription Factor Expression and Permeability of the Blood Brain Barrier Associated With Cardiopulmonary Bypass in Lambs

Marco Cavaglia, MDa, Shivaprakash G. Seshadri, MChb, James E. Marchand, PhDc,d, Carrie L. Ochocki, CCPb, Roger B. B. Mee, MB, ChBb, Paula M. Bokesch, MDc,d,*

a Department of Anesthesia Research, The Cleveland Clinic Foundation, Cleveland, Ohio, USA
b Department of Pediatric and Congenital Heart Surgery, The Cleveland Clinic Foundation, Cleveland, Ohio, USA
c Anesthesia Research, Department of Anesthesiology, Boston, Massachusetts, USA
d Tufts-New England Medical Center and Tufts University School of Medicine, Boston, Massachusetts, and the Department of Anesthesiology, Emory School of Medicine, Atlanta, Georgia, USA

Accepted for publication April 12, 2004.

* Address reprint requests to Dr Bokesch, Associate Professor, Department of Anesthesia, Children's Healthcare of Atlanta at Egleston, 1405 Clifton Rd NE, Atlanta, GA 30322, USA
paula_bokesch{at}emoryhealthcare.org


   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
BACKGROUND: The pathophysiology of neurocognitive dysfunction and developmental delay after cardiopulmonary bypass (CPB) in infants is not known. It is known that head trauma, stroke, and seizures cause dysfunction of the blood brain barrier (BBB) that is associated with increased inducible transcription factor gene expression in the cells of the barrier. The purpose of this study was to determine the effects of CPB and hypothermic circulatory arrest on expression of the transcription factor FOS and the function of the BBB in an infant animal model.

METHODS: Infant lambs (n = 36; 10–12 days) were exposed to 0, 15, 30, 60, or 120 minutes of normothermic (38°C) CPB or 2 hours of hypothermic circulatory arrest at 16°C. After terminating bypass 15 animals had their brains perfusion-fixed and removed for immunohistochemical analysis of expression of the transcription factor FOS. The other animals were perfused with fluorescent albumin to visualize the brain microvasculature. Brain sections were analyzed with a laser scanning confocal microscope.

RESULTS: Control animals (n = 6, sham operated and cannulated) exhibited normal vasculature with negligible leakage and no FOS protein expression in neurons or endothelial cells anywhere in the brain. Significant FOS expression in barrier-associated structures including the blood vessels, choroid plexus, and ependyma but not neurons occurred at all times on bypass. CPB caused leakage of fluorescent albumin from blood vessels in all animals. Two hours of normothermic CPB (n = 4) caused significant (p < 0.01) leakage in the cerebellum, cortex, hippocampus, and corpus callosum. Animals exposed to circulatory arrest experienced severe leakage throughout the brain (p < 0.001) and FOS expression in all cells.

CONCLUSIONS: These experiments indicate that the BBB is dysfunctional after all time points on normothermic CPB, BBB dysfunction is worsened by hypothermic circulatory arrest, and BBB dysfunction is associated with intense molecular activity within the barrier-forming cells. Dysfunction of the BBB may contribute to neurologic complications after heart surgery.


   Introduction
Top
 Abstract
 Introduction
Material and Methods
 Results
 Comment
 Acknowledgments
 References
 
Neurocognitive dysfunction immediately after cardiopulmonary bypass (CPB) is a well-known complication of cardiac surgery. Although cerebral injury after heart surgery was reported in 1954, recent articles have increased awareness and concern [1, 2]. Developmental abnormalities occur in 25%–30% of infants after heart surgery, in particular speech and motor dysfunction [3–5]. The etiology of brain injury is not known, although hypoperfusion, microemboli, ischemia/reperfusion, and inflammation are believed to be involved.

Magnetic resonance imaging studies performed on both adults and infants immediately after either normothermic or hypothermic CPB illustrate swelling of the brain [6–8]. Some of these studies have correlated magnetic resonance images of edema with neuropsychological changes [8] and histopathological changes in the brain after CPB [9]. Specialized endothelial cells in blood vessels within the brain parenchyma form the blood brain barrier (BBB). Previous studies have not been able to demonstrate BBB dysfunction and leakage of serum proteins within the brain from normothermic or hypothermic nonpulsatile CPB [10–13]. Using whole brain homogenates Gillinov and associates reported that the BBB was not compromised in an animal model of CPB [14].

The protooncogene c-fos (cellular-feline osteogenic sarcoma) is an inducible transcription factor (ITF) that belongs to the activating protein-1 (AP-1) family. Transcription factors are a family of inducible genes that respond within a short time (eg, minutes) after the occurrence of a stimulus. The transcribed m-RNA of the c-fos gene translates the protein FOS that binds to DNA and initiates the transcription and/or repression of other genes. ITFs are the master regulators of every cell's development, function, and response to environmental stimuli. A number of transcription factors have been identified, but c-fos is one of the most extensively studied. The rapid and intense induction of c-fos mRNA expression with the appearance of intranuclear translated FOS protein has proved to be an effective tool with regard to detecting increased intracellular activity and is considered to be an early marker of cellular activation and stress [15]. The expression of c-fos is rapidly induced in the central nervous system after seizures, trauma, and ischemia [16]. FOS expression in the cells forming the BBB is an intermediate step for signal transduction between the blood and the brain. Previously we have illustrated that FOS protein appears in ischemic areas containing degenerating neurons with a temporal and spatial expression pattern that coincides with cell death after CPB and hypothermic circulatory arrest (HCA) [17].

A recently developed microangiographic technique based on the injection of fluorescent compounds enables visualization of the entire brain microvasculature with a confocal microscope [18]. Simultaneous observation of functional changes in BBB permeability after ischemia or hypoxia is possible using fluorescent albumin, because albumin does not extravasate albuminally (from the blood vessel into the brain parenchyma) if the BBB is intact.

In the following experiments with an animal model of CPB we investigated the integrity of the BBB using fluorescent tracer methodology and the molecular activity of the cells comprising the BBB using in situ immunohistochemistry.


   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
Results
 Comment
 Acknowledgments
 References
 
All experiments were conducted in accordance with the guidelines set forth by the U.S. Dept. of Health and Human Services, Public Health Service, National Institutes of Health (Bethesda, MD) [19]. All protocols were approved by the Animal Research Committee of the Cleveland Clinic Foundation and Tufts University School of Medicine.

Study I: BBB FOS Immunohistochemistry
Cardiopulmonary Bypass
Fifteen infant lambs were anesthetized by mask with isoflurane, intubated, and mechanically ventilated with 2% isoflurane in 100% oxygen. Mean arterial pressure was maintained at 45–55 mm Hg with isoflurane. Pancuronium, 0.1 mg/kg intravenously (IV), was administered once for muscle relaxation before incision. Monitoring included a 5-lead electrocardiogram, ear pulse oximetry, and rectal and nasopharyngeal temperatures. Ventilation was adjusted to maintain the PaCO2 at 35–45 mm Hg (Table 1).


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  		Table 1. Hemodynamic and Physiologic Data (Preoperative Data Obtained Before Onset of CPB, CPB Data Obtained Before Terminating Bypass, Postoperative Data Obtained After Terminating CPB at Normothermia)

 
A percutaneous 20-G catheter was inserted into a femoral artery to monitor arterial blood pressure, arterial blood gases, and electrolytes. Another percutaneous 20-G catheter was placed in a foreleg vein to infuse lactated Ringers solution at 4 ml · kg–1 · hr–1.

Through a median sternotomy the right atrium and ascending aorta were cannulated with 24-F venous and 12-F arterial catheters ([Polystan] Polystan A/S, Vaerløse, Denmark; [Argyle] Kendall LTP, Chicopee, MA), respectively. The ductus arteriosus was ligated. The CPB circuit included a sterile Capiox SX-10 membrane oxygenator (Terumo Corp, Tokyo, Japan), Capiox pediatric arterial line filter (Terumo Corp, Tokyo, Japan), and Stockert roller pump (Stockert Instrumente, München, Germany). Isoflurane (1%–2%) was administered through the CPB circuit to maintain mean arterial blood pressure at 45–55 mm Hg. The pump prime consisted of 500 ml of fresh whole sheep blood, 25 mEq of sodium bicarbonate, 300 U of heparin, and 300 mg of CaCl2. Pump flows were 100–150 ml · kg–1 · min–1. Heparin, 300 U/kg IV, was administered before CPB. The activated clotting time (maintained > 500 seconds on bypass) was monitored with a Hemochron 400 (International Technidyne Corp, Edam, NJ).

Control animals (n = 3) were sham operated and cannulated but did not undergo CPB. The study lambs (n = 12) were placed on normothermic (38°C) CPB for 30, 60, or 120 minutes or exposed to 120 minutes of HCA at 16°–18°C, nasopharyngeal, as previously described [20]. Animals exposed to HCA were cooled for more than 34.6 ± 7.9 minutes using pH-stat strategy and had their heads packed in ice. After the circulatory arrest the animals were rewarmed on bypass for more than 30–45 minutes to 38°C using {alpha}-stat strategy and pump flows of 100–150 ml · kg–1 · min–1. Temperature gradients between the aortic and venous lines did not exceed 8°C during cooling and rewarming. The minimum inflow cooling temperature was 12°C and the maximum inflow warming temperature was 38.5°C. We intentionally avoided 15 minutes of normothermic CPB in this set of experiments because translation of FOS protein usually requires 30–45 minutes at normothermia. Approximately 30 minutes after rewarming and terminating CPB, the animals were given a bolus of potassium chloride causing them to expire. The brains were perfusion-fixed through the aortic cannula with 1000 ml chilled heparinized saline, followed by 1000 ml chilled 4% paraformaldehyde/0.1 mol/L phosphate buffer (PB), and removed for immunohistochemical analyses as previously described [17].

Immunohistochemistry
Immunohistochemistry analyses were performed to visualize and quantitate intranuclear translated FOS protein as previously described [17, 20, 21]. The 12-µm coronal sections from the cerebral cortex, dorsal hippocampus, corpus callosum, and cerebellum were pretreated with 0.1% H2O2 in methanol for 20 minutes. Slide mounted sections were incubated at 4°C overnight with a rabbit primary antibody specific for FOS protein (Santa Cruz Biotechnology, Inc, Santa Cruz, CA). The sections were incubated with biotinylated goat antirabbit serum and processed by the avidin-biotin-peroxidase method (Vector, Burlingame, CA) using diaminobenzidine as the peroxidase substrate. Mounted and coverslipped tissue sections were examined under a Zeiss microscope at 20x magnification. Intranuclear localization of black-reaction product indicated the presence of FOS protein. A blinded observer quantified the number of cells with intranuclear FOS immunoreactivity within the endothelial cells of the choroid plexus, blood vessels, and ependyma. Two 20x fields were counted for each region and averaged.

Study II: BBB Permeability With Fluorescent Albumin
Twenty-two infant lambs underwent the same experimental protocol described above. Control animals (n = 3) were sham operated and cannulated but did not undergo CPB. Study animals were placed on CPB for 15 (n = 4), 30 (n = 4), 60 (n = 3), or 120 (n = 4) minutes at normothermia (36°–38°C). Animals were randomly assigned to the duration of CPB on the morning of the experiment. Another group of animals (n = 4) were cooled to 16°–18°C and HCA was maintained for 120 minutes. All animals had 4.5F catheters placed directly in the left atrium through a purse string suture in the appendage while on CPB for fluorescein isothiocyanate (FITC)-albumin infusion. After HCA, CPB was restarted and the animals were rewarmed and weaned from CPB at 38°C. No inotropic agents other than calcium chloride were given as indicated from the electrolytes measured with the arterial blood gas samples.

Fluorescent albumin was prepared by reconstituting 500 mg of bovine desiccate albumin-FITC (FITC, Sigma A-9771, MW 69 KD 12 mol/mole albumin [Sigma Aldrich Co, St. Louis, MO]) in 50 ml of PB saline (0.1 mol/L PBS) lacking magnesium and calcium ions. The FITC solution was stirred at room temperature in the dark for 5 minutes before injection. Within 5 minutes after terminating CPB at normothermia all animals received FITC albumin, 10 ml/kg, for more than 5 minutes through the left atrial catheter. The left atrium was used in all animals to obtain mixing within the heart and as normal a distribution of cardiac output as possible.

Immediately (within 5 minutes) after infusing the FITC albumin, all animals were intravenously given potassium chloride causing them to expire. The aorta was cross-clamped before administering the potassium chloride to avoid any perfusion of the brain with potassium. The brains were removed and fixed for 72 hours in 10% formalin solution and then placed in 30% sucrose for 5 days. Coronal sections measuring 100 µm were prepared and all nuclei were counterstained with 4'-6-diamidine-2-phenylindole (DAPI).

A person blinded to the protocol analyzed the brain sections with a Leica TCS-SP spectrophotometric laser scanning confocal microscope (Leica Microsystems, Heidelberg, Germany). DAPI-stained nuclei identified specific (cerebral cortex lamina V–VII, dorsal hippocampus, corpus callosum, cerebellum granular layer) regions within the brain. A laser beam at 488 µm excited the green (FITC-albumin) fluorochromes and emission was detected with a photomultiplier tube (PMT) through a 522 µm filter. Laser intensity was set at 45% of laser power and black levels were zero for all data acquisition. PMT gain ranged from 920–980. Because the size of the fluorescent spots in a 2D image depends on the laser power, the pinhole, zoom, focus, gain, and duration of sampling time were fixed within the same section during the acquisition of data. For each brain region examined a median filter (2–3 pixel radius) was applied to reduce background noise and the threshold was established to produce a binary region of interest with vessels assigned a pixel intensity of 255 and background pixel intensity was set at zero. Because each brain slice may exhibit uneven thickness the threshold was based on maximizing the surface vessels (brightest pixels) and removing the contribution of vessels buried deeper within the slice.

Sections were scanned in 2048 x 2048 pixel format in the X–Y direction using an 8-frame scan average. Images measuring 4 mm2 were imported into Adobe Photoshop, version 5.5 (Adobe Systems Inc, San Jose, CA) for analysis. Analysis of brightness was performed using a marquee tool and applying filters from the Image Processing Toolkit, version 3.0 (Reindeer Games, Inc, Ashville, NC).

BBB leakage was quantified by visualization of albumin extravasation in the cortex, cerebellum, hippocampus, and corpus callosum. For each region of interest, the pixel intensity was measured using a gray scale of 0–255, where 0 corresponds to black and 255 corresponds to white after conversion to gray scale. We arbitrarily developed a mean pixel intensity range scale consisting of five different intervals in increments of 50.

Statistical Analyses
All values are expressed as the mean ± the standard deviation. Statistical comparisons between groups were assessed using one-way analysis of variance (ANOVA) with the Bonferroni correction. Immunohistochemical data were evaluated for the treatment groups using ANOVA. Study groups were compared using Sigma Stat (Jandel Scientific, San Rafael, CA). A p value less than 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Material and Methods
 Results
Comment
 Acknowledgments
 References
 
All animals survived the experimental protocol. The physiologic and hemodynamic data are presented in Table 1. There were no differences among study groups in mean arterial pressure, arterial blood gases, hematocrit, or electrolytes before, during, or after CPB. One animal in the 15-minute CPB group suffered a difficult venous cannulation and a mean blood pressure of 20–25 mm Hg for 5 minutes. The hemodynamic and microscopic data of this animal were excluded.

General anesthesia for 2 hours in sham-operated animals did not induce FOS protein expression in any cells in the brain (Fig 1). After 30, 60, and 120 minutes of CPB there was significant (p < 0.001) intranuclear FOS protein expression in the endothelial cells of the blood vessels, choroid plexus, and ependyma when compared with the control lambs (Figs 1, 2). There was however no FOS protein in neurons after CPB. After 120 minutes of HCA there was significant FOS protein in both neurons and endothelial cells (Fig 1). There was no statistically significant difference in the number of FOS-positive endothelial cells among the groups exposed to CPB or HCA (Fig 2).



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  		Fig 1. FOS protein expression in endothelial cells of the blood brain barrier, choroid plexus, and ependyma. (A) Control animal after 2 hours of general anesthesia, (B) FOS protein in endothelial cells of the choroid plexus and blood vessel (BV) after 30 minutes normothermic cardiopulmonary bypass (CPB), (C) after 120 minutes CPB, and (D) after 2 hours of hypothermic circulatory arrest. Bar = 50 µM. (Ep = ependyma.)

 


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  		Fig 2. FOS expression in endothelial cells of blood vessels, choroid plexus, and ependyma. The number of FOS-positive nuclei per 20x field. All time points after normothermic cardiopulmonary bypass (CPB) and hypothermic circulatory arrest (HCA) indicated significant (p < 0.001) FOS protein expression among the CPB- and HCA-treated animals.

 
The FITC-albumin injection consistently filled the intravascular compartments of the brain. Large penetrating vessels (> 50 µm) were observed perpendicular to the pial surface, whereas smaller vessels (< 20 µm) occupied all boundaries of the parenchymal space. Cellular nuclei stained red (Fig 3) from DAPI under ultraviolet fluorescence. Regions of impaired BBB function were detected as areas with abnormal extravasation of the fluorescent dye and were observed as yellow discolorations around the blood vessels in Figures 4A and 4B.



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  		Fig 3. General anesthesia does not disturb the blood brain barrier. 20x magnification of 100 µm coronal section of lamb cerebral cortex after 2 hours of isoflurane anesthesia. Animals were sham operated and cannulated but did not undergo cardiopulmonary bypass. Intravascular albumin displays the vasculature of the frontal cerebral cortex without leakage. 4'-6-diamidine-2-phenylindole-stained nuclei appear red.

 


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  		Fig 4. Normothermic cardiopulmonary bypass (CPB) causes increased permeability of the blood brain barrier. (A) 20x magnification of 100 µm coronal section of lamb frontal cerebral cortex immediately after terminating 2 hours of normothermic CPB. (B) 200x magnification of the same region of the cortex indicating extravasation of dye (yellow) from neocortical vessels. 4'-6-diamidine-2-phenylindole-stained nuclei appear red.

 
Animals under general anesthesia for 2 hours experienced normal vasculature and no observable leakage of the BBB anywhere in the brain. The mean pixel intensity/mm2 was 54.5 ± 6.8 in all areas of the brain and is considered background pixel intensity (Fig 5, control).



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  		Fig 5. Blood brain barrier leakage. Animals exposed to normothermic cardiopulmonary bypass (CPB) for 15, 30, 60, or 120 minutes experienced significant leakage (p < 0.05 versus control) in the cerebral cortex and corpus callosum. Leakage in the cerebellum and hippocampus was significant after 60 and 30 minutes of CPB, respectively.

 
Animals exposed to normothermic CPB experienced increasing leakage of FITC-albumin (Fig 5). After 2 hours of normothermic CPB animals experienced significant leakage of the BBB in the cerebellum (p = 0.0003), cortex (p = 0.0007), hippocampus (p = 0.003), and corpus callosum (p = 0.009) when compared with control animals (Fig 5). There was no significant leakage of the BBB above background in the hippocampus after 15 minutes of normothermic CPB or after 15 and 30 minutes in the cerebellum (Fig 5).

Animals exposed to 2 hours of HCA experienced severe leakage of the BBB in all areas of the brain. HCA animals also experienced significantly greater leakage of the BBB than animals with 2 hours of CPB in the cerebellum (pixel intensity/mm2 238 ± 22 vs 159 ± 33; p = 0.009), cortex (222 ± 28 vs 143 ± 23; p = 0.004), hippocampus (172 ± 20 vs 86 ± 16; p = 0.0002), and corpus callosum (205 ± 25 vs 132 ± 20; p = 0.003; data not shown).


   Comment
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
Acknowledgments
 References
 
These experiments indicate that CPB increases the permeability of the BBB and increases the expression of the ITF, c-fos, in the cells comprising the BBB in infant lambs (Figs 1, 2). The amount of leakage increases with the duration of CPB (Fig 5). HCA causes extensive leakage of the BBB throughout the brain and FOS expression in neurons as well as endothelial cells. During CPB leakage of the BBB could also expose neurons to various cytotoxic substances produced during extracorporeal circulation that would normally be confined to the vasculature.

FOS protein functions as a transcriptional regulator at the AP-1 binding site on DNA inducing the transcription or suppression of effector genes. Whereas FOS was not expressed in the control animals it is consistently upregulated in the endothelial cells of the BBB, choroid plexus, and ependyma in all experiments using CPB, but not in neurons (except with HCA). It is not clear from these experiments which effector genes are subsequently transcribed or suppressed.

We selected 2 hours of HCA because of previous work performed in our laboratory indicating that this time was required to observe neuronal degeneration [17, 21]. We previously reported that translation of FOS protein is significantly impaired in neurons after 2 hours of HCA [17]. In contrast the present experiments demonstrate that FOS protein translation in endothelial cells in the same brain regions is not impaired after 2 hours of HCA. There is no significant difference in the amount of FOS protein expression in the BBB structures with either CPB or HCA (Figs 1, 2).

The BBB maintains homeostasis of the neuronal environment by limiting blood-to-brain diffusion of hydrophilic molecules, restricting penetration to lipophilic substances capable of directly traversing endothelial membranes, and regulating the passage of substances such as amino acids and glucose that contain specific membrane carriers. After brain ischemia BBB disruption leads to the extravascular leakage of plasma proteins and other solutes resulting in an imbalance with osmotic forces that draws excess water into the tissue [22]. Tissue swelling ensues within the rigid confines of the skull potentially elevating intracranial pressure leading to further reductions in cerebral blood flow.

BBB disruption is thought to play a critical role in the pathophysiology of ischemia/reperfusion injury. Transient occlusion of the middle cerebral artery in rodents consistently produces evidence of BBB disruption 3–4 hours after reperfusion [23, 24]. We have also previously reported that BBB leakage of FITC-albumin occurs 3–4 hours after cerebral ischemia in rodents [25]. In the present study BBB disruption is observed immediately after terminating CPB with restoration of normal circulation rather than 3–4 hours after reperfusion. The amount of leakage seems to increase with the duration of CPB, but did not achieve statistical significance. Based on this evidence it seems that the processes inducing BBB leakage occur with the onset and duration of CPB. The early onset of BBB dysfunction, observed after only 15 minutes of CPB, implies that this mechanism may be a primary insult to the brain.

The immunohistochemistry data supports the theory that endothelial cell-mediated processes involving ITF expression are associated with the dysfunction of the BBB. The ITF c-fos is transcribed rapidly in the central nervous system in response to cellular stress such as hypoxia, ischemia, trauma, and hypoperfusion. Whereas transcription of c-fos is immediate, the translation of FOS protein usually requires 30 minutes or longer. The presence of intranuclear FOS protein in the endothelial cells of the BBB after only 30 minutes of normothermic CPB indicates that the transcription of c-fos occurs with the onset and duration of CPB. Although these results do not exclude reperfusion-injury mechanisms after CPB, the initial stress to the brain occurs during CPB.

Gillinov and associates reported that the BBB was not compromised in an animal model of CPB at 28°C. However they used a homogenate of brain areas to quantitate the volume of radioactive carbon 14-aminoisobutyric acid [14]. Their method cannot differentiate intravascular from extravascular tracer and only indicates that the total volume of tracer in the brain is not different from control animals. In addition it is possible that their moderate hypothermic CPB model prevents the BBB changes described in our normothermic model of CPB. Using the peroxidase–antiperoxidase technique of immunocytochemical staining, Laursen and associates reported minute foci of extravasated serum proteins in brain sections after 3 hours of normothermic pulsatile CPB, but not with nonpulsatile CPB [10]. Using the same method Waaben and associates reported that 2 hours of normothermic CPB did not change the cerebrovascular permeability to serum proteins [12]. The present study used a fluorescent tracer methodology that quantifies minor degrees in BBB opening in situ that may not be detected with staining techniques. Our results, which clearly demonstrate BBB leakage after only 15 and 30 minutes of normothermic CPB, may reflect a more sensitive method of detection. Alternatively the present study was performed in infant lambs unlike the older animals used in the above experiments. The immature brain may be more vulnerable to BBB dysfunction.

There are several limitations regarding the interpretation of data from these experiments. First we were unable to assess neuropsychological damage in this model. Sheep are not likely to display cognitive function let alone subtle dysfunction. Therefore we cannot conclude that the leakage of the BBB is causing neurocognitive dysfunction. Second we observed one time point only immediately after terminating CPB when "normal" pulsatile blood flow to the brain was restored. At this time point the BBB is clearly dysfunctional. Additional experiments are required to determine how long the BBB is hyperpermeable after the termination of CPB and whether moderate hypothermia can attenuate this response. Mild hypothermia (30°–32°C) has been reported to maintain the impermeability of the BBB and prevent the development of ischemia-induced vasogenic brain edema [26, 27]. Hypothermia to 28°C has also been effective at preventing the perivascular swelling of the endfeet of astrocytes observed in pig brains with normothermic CPB [11]. Finally the present experiments were performed in infant animal models and the results may differ in adult animals. Further experiments in adult animals are required to determine if the BBB is as dysfunctional as we observed in these experiments.

We can only speculate on the mechanisms whereby CPB induces the expression of FOS in endothelial cells and increases permeability of the BBB. These mechanisms may include complement activation and inflammation secondary to blood contact with the CPB circuit, nonpulsatile flow and hypoperfusion of the brain, NO-mediated relaxation of the tight junctions, and/or gaseous microemboli [28–32]. Cytokines have been indicated to increase vascular permeability [33] and open the BBB [34]. Endothelial cell activation is an intermediate step for signal transduction between the blood and the brain. Unlike other target organs the brain is protected from access by signaling molecules such as complement and cytokines that do not readily cross the BBB. However complement and cytokine receptors have been demonstrated on the endothelial cells of the BBB [35]. Through these receptors cytokines stimulate the induction of c-fos mRNA and FOS protein. Molecules generated by FOS within these activated barrier cells provide signals to neighboring neurons. In this manner BBB cells can transduce peripheral cytokine signals to neurons within the brain [36].

The present study suggests that the initial underlying pathophysiology of the brain edema observed on MRI after CPB may be dysfunction of the BBB. In brain tissue increased fluid can occur from increased passage of water and solutes across the BBB, increased passage from brain cells into the brain interstitial space, or decreased uptake from the brain interstitial spaces into brain cells [37]. Although the present study confirms that there is increased passage of albumin across the BBB, we did not determine whether increased production or decreased absorption of cerebrospinal fluid (CSF) is also occurring. Neither can we rule out whether elevated venous pressure in the superior vena cava (not measured in the present study) is impairing CSF absorption as has been suggested in patients during and after heart surgery using CPB [8]. If the edema is severe, as after HCA, it is likely that neurons will suffer further ischemic insult and die from excitotoxic mechanisms as we previously reported [17, 21]. On the other hand mild leakage of the BBB in the cortex, hippocampus, corpus callosum, and cerebellum after normothermic CPB will cause more subtle and possibly transient dysfunction or no dysfunction. The clinical observation of motor and speech abnormalities in the developing brains of infants after CPB [5] may be associated with the leakage we observed in the cerebellum, which was particularly vulnerable in this infant animal model after HCA.

In summary, both normothermic CPB and HCA cause increased FOS expression in the endothelial cells of the brain and BBB dysfunction in infant lambs. BBB leakage seems to be an early initial insult of CPB that may contribute to the brain edema observed clinically and in animal models after CPB. BBB leakage may also contribute to the neuropsychological and motor dysfunction observed after CPB. Stabilizing the BBB, possibly through inhibition of complement, cytokines, or guanylate cyclase, and NO release, may improve the outcome after heart surgery.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
References
 
This work was supported by The American Heart Association (Grant No. 9951512V). Research was performed at the Department of Anesthesia Research and The Department of Pediatric and Congenital Heart Surgery at the Cleveland Clinic Foundation and research on anesthesia was performed at Tufts University School of Medicine. Doctor Paula Bokesch was the Principal Investigator.


   References
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Acknowledgments
 References
 

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