Anesth Analg 2005;101:1465-1469
© 2005 International Anesthesia Research Society
doi: 10.1213/01.ANE.0000180193.29655.6A
CRITICAL CARE AND TRAUMA
Slight Increase of Serum S-100B During Porcine Endotoxemic Shock May Indicate Blood-Brain Barrier Damage
Anders Larsson, MD, PhD*,
Miklós Lipcsey, MD
,
Jan Sjölin, MD, PhD*,
Lars-Olof Hansson, MD, PhD*, and
Mats B. Eriksson, MD, PhD
Departments of *Medical Sciences and Surgical Sciences, Uppsala University Hospital, Sweden
Address correspondence and reprint requests to Anders Larsson, MD, PhD, Department of Medical Sciences, University Hospital S-751 85 Uppsala, Sweden. Address e-mail to anders.larsson{at}akademiska.se.
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Abstract
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Septic shock is a condition that affects many organs, but little is known about the effects on the central nervous system. S-100B, an acidic low molecular weight protein, has attracted considerable interest as a marker for brain damage and disintegration of the blood-brain barrier. It is released into the cerebrospinal fluid and blood from brain tissue after brain damage. We studied S-100B in a porcine model of endotoxemic shock that resembles human Gram-negative septic shock. Ten piglets received IV endotoxin, and plasma samples were collected before the endotoxin infusion and each hour (1–6 h) during the endotoxin infusion. S-100B was measured by sandwich enzyme-linked immunosorbent assay. Low levels of plasma S-100B were detected, but there was a significant increase in S-100B during Hours 1–5 in comparison with the 0 values. We determined that endotoxemia causes a very small but significant increase in the levels of the widely used brain damage marker serum S-100B. However, it cannot be excluded that the increase in S-100B could be caused by release from organs other than the brain.
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Introduction
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S-100B is a 20-kDa protein belonging to the S-100/calmodulin/troponin C superfamily of calcium-binding proteins. S-100 was originally isolated from the human brain and is considered a glial-cell-specific protein (1). Today, 20 monomers of the S-100 family have been identified based on structural and functional similarities (2,3). Most of the S-100 proteins exist as dimers and are expressed in a cell-specific manner. Two of the S-100 monomers, designated S-100A1 and S-100B (4), are highly conserved between species and are found as homo- (BB) and hetero-dimers (A1B) in the cytoplasm of central nervous system (CNS) glial cells and in certain peripheral cells, e.g., Schwann cells, melanocytes, adipocytes, and chondrocytes (5). S-100A1B and S-100BB are also present in malignant tissues, most notably in melanoma and, to a lesser extent, in glioma, thyroid cell carcinoma, and renal cell carcinoma (2). Determina- tion of S-100B in serum has been shown to be clinically useful for prognosis and treatment monitoring of patients diagnosed with malignant melanoma (6–9). S-100B has been considered to be a marker of the blood-brain barrier and has thus been used in the management of patients with brain damage from causes, such as traumatic head injury, perinatal asphyxia, cardiac arrest, cardiac surgery, and stroke (10–13). S-100B is often considered as a specific brain damage marker (astroglial and Schwann cells), but it may also be increased because of damage to other tissues (14). S-100B has previously been measured in pigs with acute encephalopathy (15,16) and during circulatory arrest and cardiopulmonary resuscitation (17,18) and has been shown to be related to coma depth, time of anoxia, and long-term outcome after cardiac arrest (19). Even a minor head injury, such as repeated heading by soccer players, results in increased S-100B levels (20).
Deterioration of neurologic function accompanies organ failure in sepsis patients (21). Patients who have sepsis with stable or supported hemodynamics and adequate oxygenation may manifest with altered mental status. Cerebral hypoperfusion, anoxia, and progressive edema of the brain have been suggested as potentially causative, but the pathogenesis remains unknown. The brain is the source of several inflammatory mediators that have an impact on cerebrovascular function. Experimental porcine endotoxemic shock has been shown to cause significant increases of intracranial pressure and increased cerebral blood volume despite arterial hypotension (22). The aim of the present investigation was to study whether the blood-brain marker S-100B, which can repeatedly be analyzed in serum samples, is increased in porcine endotoxemic shock and, if so, whether this is related to changes in the systemic vascular permeability.
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Methods
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The study included 14 domestic breed piglets of both sexes weighing between 24 and 30 kg (average 27 kg). Ten piglets received an endotoxin infusion, and four piglets served as a control group. All animals were between 12 and 14 wk old and apparently healthy. All piglets were handled according to the guidelines of the Swedish National Board for Laboratory Animals and the European Convention on Animal Care. The experiment was approved by the Animal Ethics Committee of the University of Uppsala, Sweden.
The study inclusion criteria included patients with no apparent preexisting diseases, Pao2 >10 kPa (75 mm Hg) in arterial blood, and a mean pulmonary artery pressure (MPAP) <2.7 kPa (20 mm Hg) at baseline and 20 min after completed preparatory procedure (see below).
General anesthesia was induced by injecting a mixture of 6 mg/kg of tilétamin-zolazepam (Zoletil forte vetTM, Virbac Laboratories, Carros, France), 2.2 mg/kg of xylazine (Rompun Vet, Bayer, Leverkusen, Germany), and 0.04 mg/kg of atropine (AtropinTM, NM Pharma, Stockholm, Sweden) IM. Anesthesia was then maintained with sodium pentobarbital (8 mg · kg–1 · h–1; Pentobarbital-NatriumTM, Apoteket, Umeå, Sweden), pancuronium bromide (0.26 mg · kg–1 · h–1; PavulonTM, Organon, Oss, The Netherlands), and morphine (0.48 mg · kg–1 · h–1, MorphineTM, Pharmacia, Uppsala, Sweden) dissolved in 2.5% glucose solution given as a continuous infusion. Also, sodium chloride infusion was given, resulting in a total fluid administration rate of 30 mL · kg–1 · h–1.
A bolus dose of 20 mg of morphine was given IV before the performance of a tracheotomy, which was performed to secure a free airway during the experiment. The animals were artificially ventilated throughout the experimental procedure (Servo 900CTM, Siemens-Elema, Stockholm, Sweden). During surgical stimulation, i.e., catheter insertion, 30% oxygen in N2O was given, after which the gas mixture was set to a fraction of inspired oxygen (Fio2) of 0.3 in medical air during the rest of the experiment. The ventilation after preparation was adjusted to yield a Paco2 between 5.0 and 5.5 kPa. The respiratory rate was 25 breaths/min, and the inspiratory-expiratory ratio was 1:3. Respirator settings were then kept constant throughout the experiment. To monitor hemodynamics and to permit blood sampling, a cervical artery was catheterized, and a central venous line and a 7F Swan-Ganz catheter (equipped with thermistor) were inserted through the internal jugular vein into the superior caval vein and the pulmonary artery, respectively. A minor vesicotomy was performed, and a urinary catheter was introduced into the bladder. A heating pad (Operatherm 200W, KanMed, Bromma, Sweden) was used to keep the animals at a constant body temperature. After preparation, there was a 20-min stabilization period before baseline values were registered and baseline blood samples were taken.
Hypodynamic endotoxemic shock was induced in 10 of the piglets with a 3-h continuous infusion of Escherichia coli endotoxin (O111:B4; Sigma Chemicals, St. Louis, MO), where at least doubling of the initial MPAP during the first hour was take as a sign of endotoxemic shock. This model has previously been described by us (23) and others (24).
S-100B was measured by a human S-100 sandwich enzyme-linked immunosorbent assay (ELISA; S100 EIA, Prod. No. 708–10; CanAg Diagnostics AB, Gothenburg, Sweden). The antibodies in the ELISA are raised against bovine S-100B and detect S-100A1B and S-100BB. S-100B is highly conserved between species, with only a single amino acid difference between human and bovine S-100B. The ELISA can thus be used to detect S-100 from different species. Hemoglobin was analyzed with an ABL 300 (Radiometer, Brønhøj, Denmark) as a marker for increased vascular permeability. A commercial sandwich ELISA was used for the detection of interleukin-6 (Quantikine porcine IL-6, P6000; R&D Systems, Minneapolis, MN). The lower detection limit was 10 pg/mL, and the assay had an intraassay coefficient of variation (CV) of <5% and total CV of <10%.
For statistical analysis, the relative changes for all values were calculated setting baseline values as zero indexes. Differences between the animals at different times were calculated by an analysis of variance test. These calculations were based on the individual values for each pig at each time of measurement. Spearman rank correlation coefficients (R) and P values were used for comparison between S-100B and hemoglobin values. A P value <0.05 was considered significant.
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Results
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The animals were comparable in physiological baseline variables. All pigs given endotoxin responded to this challenge with a marked increase in MPAP and a continuous deterioration in circulatory and respiratory variables (Table 1). One pig died after the first hour of the experiment.
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Table 1. Physiologic Variables Expressed as Absolute Values During Endotoxin Infusion (mean ± sd). Interleukin-6 is expressed as geometric mean ± sd.
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At baseline (0 h), all animals had low S-100B values. S-100B increased during the first 3–4 h of the experiment and decreased afterward to return to baseline values (Figs. 1 and 2). Both relative and absolute S-100B values were increased from 1 h to 5 h in comparison with 0 h (P < 0.05). There was no significant difference between 0-h and 6-h values. No increase in S-100B values was detected in any of the animals in the control group (results not shown).
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Figure 1. S-100B levels in percentage of baseline value (0 value) in individual animals. The results are presented as median, quartiles, and range.
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Figure 2. S-100B levels in individual animals. The results are presented as median, quartiles, and range.
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Hemoglobin increased from baseline to 3 h as a sign of hemoconcentration and increased vascular permeability. From 4 h to 6 h, there was a decrease in hemoglobin concentration (Fig. 3). The hemoglobin values at the end of the experiment (6 h) were not significantly different from the baseline values. The change in hemoglobin over time was similar to the changes observed for S-100B, except that the amplitude of the changes was less pronounced for hemoglobin. There was also a very strong Spearman rank correlation between the relative hemoglobin and S-100B values (RS = 0.57; P = 0.0001).
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Figure 3. Hemoglobin levels in percentage of baseline value (0 value) in individual animals. The results are presented as median, quartiles, and range.
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Discussion
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Human septic illness is frequently accompanied by abnormal cerebral manifestations, where the severity of brain dysfunction correlates with fatal outcome (25). In various porcine models of endotoxemia, expression of inflammatory mediators has been detected in the brain (26), and cerebrovascular dysfunction is associated with maldistribution of the cerebral blood flow (21). The exact mechanism by which encephalopathy secondary to sepsis ensues is not known, but amino acid derangements, blood-brain barrier disruption, abnormal neurotransmitters, and direct CNS effects may all contribute to this effect on the CNS (27). Nonlipophilic substances, and especially charged proteins, such as S-100B, have a poor ability to penetrate the tight junctions of the blood-brain barrier. When this barrier is disrupted, its penetrability is highly increased. Interestingly, not only patients suffering from infections that primarily affect the brain, but also those with severe, primarily extracerebral infectious diseases, had increased levels of S-100B, although the levels were less frequently increased and less often expressed among the patients with extracerebral disease (28).
There is no specific test available for porcine S-100B, but S-100B is highly conserved between species, with only a single amino acid substitution. The test used in the study uses antibodies directed against bovine S-100B and can be used to detect S-100B from different mammalian species. It has previously been shown that porcine S-100B can be detected by assays intended for human use (15,16). Because serum S-100B is a marker for brain and blood-brain barrier damage, serum samples may be an interesting alternative to cerebrospinal samples in the assessment of cerebral injury in endotoxemic shock. If similar results are found in patients with septic shock, analysis of serum S-100B may offer an option to monitor organ dysfunction of the CNS in this patient group. However, this requires further investigation.
In this model of porcine endotoxemia leading to infrequent mortality but frequent morbidity, a small but significant increase in S-100B was found. No increase in S-100B was detected in the control group, indicating that the S-100B increase was caused by the endotoxemia and not to the surgical procedures or the anesthesia. This is in agreement with previous reports that showed that anesthesia did not cause an increase in S-100B (15). Furthermore, our anesthetic procedure is based on continuous infusion of pentobarbital, which is frequently used to reduce increased intracranial pressure (29). Change in plasma hemoglobin concentration was used as a marker for increased vascular permeability. We found a strong correlation between the relative hemoglobin and S-100B values. The correlation is in agreement with a general increase in vascular permeability. At 3 h, the mean base excess was –3 mmol/L, excluding the possibility that severe ischemic damage was responsible for the observed increase in S-100B. Thus, we propose that the endotoxin effect on glial cellular integrity and blood-brain barrier disruption was the main cause of this increase, especially because the kinetic of the S-100B increase was similar to the increase in hemoglobin that was used as a marker for increased permeability.
In conclusion, the increase of S-100B found in this study indicates that there is minor blood-brain barrier damage during porcine endotoxemic shock and that this increase is correlated to the degree of systemic vascular permeability. However, we cannot exclude the possibility that other cells could be the source of the S-100B increase. S-100B could have been a potential marker for studies of CNS effects during septicemia, but further studies are required, e.g., to elucidate the effects of cerebral blood flow on S-100B release from the brain to the blood circulation.
The S-100B assay was generously provided by CanAg Diagnostics AB, Gothenburg, Sweden. The technical assistance of Anders Nordgren is greatly appreciated.
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Footnotes
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Supported, in part, by grants from the Laerdal Foundation for Acute Medicine, Stavanger, Norway.
Accepted for publication April 20, 2005.
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Abstract
Introduction
