S 100 B, a calcium-binding protein (1) present in high concentrations in astroglial and Schwann cells (2), is an acknowledged marker of brain damage (3–8). Furthermore, S 100 B increases have been found following global cerebral ischemia after cardiac arrest (9), hypothermic circulatory arrest (10), during cardiopulmonary bypass surgery (11), in stroke (12), and in aneurysmal subarachnoid hemorrhage (13).
Interestingly, recent publications report that the S 100 B increase in the early phase after cardiac surgery is not due to release of S 100 B from the brain alone, but also from tissue outside the brain (14). This is in line with other publications that are questioning the S 100 B increase in serum during cardiopulmonary bypass as a sign of brain injury (15). After trauma, S 100 B increases have also been reported without brain injury (16). This is in accordance with our own findings in a prospective clinical multicenter study (17). In this study, we measured S 100 B in patients with hemorrhagic shock and one of three different patterns of trauma: isolated traumatic brain injury, traumatic brain injury with multiple trauma, and multiple trauma without traumatic brain injury. Surprisingly, we found an initial posttraumatic S 100 B increase in all patients with multiple trauma and hemorrhagic shock, including those without traumatic brain injury. After this initial increase, S 100 B decreased to normal values after 24–48 h. On the basis of these findings, we are now attempting to answer the following questions: What causes the initial posttraumatic S 100 B increase in the absence of cerebral trauma? Does extracerebral soft tissue injury cause it? Does hemorrhagic shock cause it and, if so, is the S 100 B increase associated with the severity of shock? In an effort to answer these questions, we conducted the following experimental study which demonstrates for the first time that S 100 B is released into serum by experimental hemorrhagic shock in rats, even in the absence of concomitant traumatic brain injury. In addition, S 100 B increase is associated with the severity of hemorrhagic shock.
Our study provides an experimental basis for further evaluating the significance of S 100 B as a diagnostic parameter for the severity of hemorrhage in severely injured patients. Our findings are relevant in the setting of multiple trauma, where S 100 B is thought to reflect the extent of injury and to predict outcome.
MATERIALS AND METHODS
The experimental protocol of the study was approved by the Animal Protocol Review Board of Vienna. The requirements defined in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (publication NIH 86-23, revised 1985) were strictly adhered to. Male Sprague-Dawley rats (n = 50, 400–440 g; Animal Research Laboratories, Himberg, Austria) were granted free access to standard laboratory chow and water during a 7-day adaptation period after delivery to our experimental unit. Before the experiment, the rats were fasted overnight with further free access to water. Anesthesia was induced with ketamine/xylazine (112/10 mg/kg i.m.) and inhalation anesthesia with spontaneous respiration was maintained with 0.5% isoflurane. The left femoral artery was cannulated with a polyethylene catheter (PE-50; Reichelt Chemie Technik, Heidelberg, Germany) and connected to a blood pressure monitor (Cardiosys, Budapest, Hungary). The rats were positioned and kept on a temperature-controlled surgical board (36°C–37°C) for the entire duration of the experiment, which began after a 20-min stabilization phase. All surgical procedures were performed under aseptic conditions.
The experimental procedure is shown in Figure 1. To achieve hemorrhagic shock, blood was withdrawn via a femoral artery catheter until the onset of shock at 30–35 mmHg mean arterial pressure (MAP). Soft tissue injury was induced by midline laparotomy performed before the onset of hemorrhage and was maintained for a duration of 30 min. MAP was maintained at 30–35 mmHg until the onset of decompensation. At this point, MAP was either increased immediately (moderate shock) by administration of Ringer's solution to a low-flow phase of 40–45 mmHg, or MAP was maintained further at 30–35 mmHg (severe shock) until 40% of the shed blood had been administered as Ringer's solution and was then increased to a low-flow phase of 40–45 mmHg. After MAP had been increased to 40–45 mmHg, this low flow phase of 40–45 mmHg was maintained for 40 min by administration of an adequate volume of Ringer's solution. Until this point in the experiment, no shed blood was readministered and no heparin was involved in vivo. Subsequently, rats were resuscitated for 60 min by administering heparinized (10 IU/mL) shed blood filtered through 5-μm filters (Sterifix; B. Braun Melsungen, Melsungen, Germany) to prevent thrombosis, and the double volume of Ringer's solution. Because the volume of shed blood did not differ between the groups at the onset of decompensation, similar volumes of heparinized blood were administered after moderate shock (Group A) and after severe shock (Group B) during resuscitation. In the rats without hemorrhagic shock (Group C), midline laparotomy was performed and maintained for a duration of 30 min, but no blood was withdrawn.
Fifty rats were randomly divided into three groups as follows: moderate hemorrhagic shock with laparotomy (Group A, n = 25), severe hemorrhagic shock with laparotomy (Group B, n = 20), and laparotomy without hemorrhagic shock (Group C, n = 5). In moderate hemorrhagic shock (Group A) and severe hemorrhagic shock (Group B), five rats were sacrificed after arterial blood samples had been obtained for measurement of S 100 B (1 mL of whole blood) and pH, base excess, and lactate (0.5 mL of whole blood) at each of the following time points: baseline, end of shock (MAP 30–35 mmHg), end of low flow (MAP 40–45 mmHg), and 3 h after resuscitation. Five rats were sacrificed 24 h after resuscitation in moderate shock (Group A) only. Because our pilot studies have shown that most of the rats exposed to the same protocol of severe shock die before 24 h, this time point was not considered for severe hemorrhagic shock (Group B) in the present experiment. In Group C (no hemorrhagic shock), blood samples were obtained at the following time points: baseline, 3 h after laparotomy (corresponding to end of shock/end of low flow), and 8 h after laparotomy (corresponding to 3 h after resuscitation). All rats in Group C were sacrificed after the last blood sample had been obtained. The reason why the rats in Groups A and B were sacrificed at each time point was that histological organ evaluation after hemorrhagic shock was required. This was the subject of a separate second experiment, which was not associated with the experiments presented here, but was performed on the same animals.
Arterial heparinized rat blood samples were then centrifuged and plasma was frozen to –72°C and stored for analysis. For S 100 B measurement in plasma, samples were analyzed with a commercially available immunoluminometric assay (LIA mat Sangtec 100; Byk-Sangtec Diagnostika, Bromma, Sweden). This is a monoclonal two-site immunoassay with a detection limit of 0.1 μg/L for S 100 B in humans. According to the manufacturer, this test has been found to be cross-reactive with rat plasma. Because the normal range for rats is not known, baseline values in rats were considered normal and all other S 100 B values were compared with baseline.
For measurement of pH, base excess, and lactate, samples were analyzed using ABL 625 System (Copenhagen, Denmark).
Data are shown as medians and 25%/75% percentiles (Q1/Q3). Differences between the two shock groups (moderate and severe shock) were tested using the Mann-Whitney U test. Differences between time points were tested using analysis of variance (ANOVA) followed by post hoc Mann-Whitney. The level of significance was set at P < 0.05.
S 100 B in plasma was significantly increased both in soft tissue injury with moderate hemorrhagic shock (Group A), and in soft tissue injury with severe hemorrhagic shock (Group B). Findings in severe hemorrhagic shock were significantly different from findings in moderate hemorrhagic shock, both regarding S 100 B and metabolic acidosis. In severe hemorrhagic shock, S 100 B almost tripled at the end of shock and increased almost 5-fold at the end of the low-flow phase (Fig. 2).
The volume of shed blood was not significantly different between the moderate and the severe hemorrhagic shock group (28.8 ± 3.2 mL/kg and 31.9 ± 5.6 mL/kg, respectively). Moreover, the volume of Ringer's solution administered during the low-flow phase was not significantly different between the moderate and the severe hemorrhagic shock group (16.7 ± 5.6 mL/kg and 17.7 ± 4.8 mL/kg, respectively).
As expected, metabolic acidosis was significantly more pronounced in the severe hemorrhagic shock group than it was in the moderate hemorrhagic shock group (Table 1). In contrast, the group with no hemorrhagic shock (Group C) showed neither metabolic acidosis nor an S 100 B increase, and S 100 B remained normal both 3 and 8 h after laparotomy (Table 2).
The general condition of the rats during the experiment can only be judged to a limited extent because most of the rats were still anesthetized when sacrificed. As pointed out earlier, five rats each were sacrificed after arterial blood samples had been obtained at each time point. Only the rats sacrificed 24 h after resuscitation (5 of 25 rats in moderate shock, Group A), emerged from anesthesia. These rats were conscious but showed little spontaneous motoric activity.
Hemorrhagic shock induced an increase of S 100 B in plasma. This S 100 B increase was significantly higher in severe than in moderate hemorrhagic shock. In contrast, soft tissue injury without hemorrhagic shock did not induce an increase of S 100 B. These experimental findings are similar to the clinical findings in our above-mentioned prospective multicenter study (17): S 100 B is increased in patients with hemorrhagic shock immediately after multiple trauma without traumatic brain injury. Considering both our experimental and clinical findings, we believe that the initial posttraumatic S 100 B increase is induced by hemorrhagic shock and not by extracerebral soft tissue injury.
The single most important factor appears to be the severity of hemorrhagic shock because the immediate posttraumatic S 100 B increase is significantly more pronounced in severe than in moderate shock, and there is no posttraumatic S 100 B increase in extracerebral soft tissue injury without hemorrhagic shock. In the clinical posttraumatic setting, this may be highly relevant. Whether the source of increased S 100 B is cerebral or extracerebral has yet to be determined. The immediate posttraumatic S 100 B increase could nevertheless be of cerebral origin, even in the absence of direct cerebral trauma. Because S 100 B is present in astroglia, S 100 B increase might be an indication of ongoing brain damage due to a decrease in cerebral perfusion during severe shock. On the one hand, using the microsphere technique in a time-fixed hemorrhagic shock model, we have found that cerebral perfusion (unlike perfusion in all other regions except the adrenal cortex) is globally increased rather than decreased during shock (unpublished data by Bahrami, S). On the other hand, although cerebral perfusion is globally increased, it might nevertheless be locally decreased in some areas of the brain (18). Thus, some areas of the brain might be hypoperfused during shock and resuscitation (19) and could thus be the source of increased S 100 B. However, this is merely a speculative assumption because we did not assess a leakage of S 100 B from the intrathecal compartment across the blood-brain barrier in our study.
In the moderate shock group, S 100 B levels remained increased 5- to 10-fold after 24 h. This in an interesting observation considering the fact that S 100 B has a half-life of approximately 2 h (1). In our opinion, the consistently increased levels could be due to an ongoing S 100 B release, commencing during hemorrhagic shock and continuing to a lesser but nevertheless clearly detectable extent throughout and even after the resuscitation phase.
According to the literature, there is no known S 100 B increase due to heparin. In contrast, serum S 100 protein levels have been shown to be reduced after using arterial line filtration and covalent-bonded heparin coating during cardiac surgery (20). In the present experiment, heparin was administered for the first time when shed blood was readministered during resuscitation after the low-flow phase. In vivo, no heparin was administered before this time point. In vitro, however, heparin plasma used for S100 B analysis was obtained for all samples including those drawn at baseline. The amount of heparin added was the same in all samples (8 units of heparin/mL whole blood) and at all time points. Therefore, the relative changes in S 100 B and the differences between moderate and severe shock could not have been due to heparin. However, the absolute values and changes in the rat remain unknown.
Clinically, the serum S 100 B increase measured posttraumatically has been attributed not only to surgically traumatized fat and muscle, but also to traumatized bone marrow (16). According to a recently published study on patients undergoing cardiac surgery, the systemic serum S 100 B increase is not due to the release of S 100 B from the brain alone, but also to the release from tissue outside the brain (14). Another recent study is questioning the interpretation that an increase in S 100 B in patients undergoing cardiopulmonary bypass reflects cerebral injury (15). In our opinion, the bone marrow of the sternum, which is traumatized in cardiac surgery, is a possible source of S 100 B outside the brain. In contrast, our laparotomy model, which mimics surgically traumatized fat and muscle but not traumatized bone marrow, was not associated with any serum S 100 B increase at all.
Our experimental model of hemorrhagic shock was meant to mimic the posttraumatic clinical setting as closely as possible. The actual condition of a trauma patient who has lost around 40% of his blood volume is variable and difficult to mimic experimentally. Several factors, including the patient's age and general medical condition and history prior to trauma, influence the actual condition of the trauma patient in hemorrhagic shock. Unlike trauma patients, experimental animals are very similar. Therefore, the clinical severity of shock is simulated by variation of the decompensation period. Moreover, resuscitation in the clinical setting is often inadequate. Our unique animal model, in which the low-flow phase corresponds to inadequate resuscitation, was developed to mimic this situation. In addition, clinical hemorrhage is always associated also with some degree of soft tissue injury. In our experiment, this extracerebral soft tissue injury was represented by laparotomy. The initial shock phase (MAP 30–35 mmHg) mimics the immediate posttraumatic setting without resuscitation. The subsequent low-flow state (MAP 40–45 mmHg) reflects the situation immediately after admission to the emergency room, during which resuscitation is still inadequate. In our preliminary pilot studies, the 6-day mortality rate was found to be 40% in moderate shock vs. 90% in severe shock. Because most animals have been found to die prior to 24 h after severe shock, we did not include the 24-h time point for the severe shock group in our experiment. A sham group without laparotomy but with anesthesia and catherization was not included in the experiment. Because laparotomy with anesthesia and catheterization did not cause an increase in S 100 B, one cannot expect that anesthesia and catherization would have done so. This observation (which we made during our pilot experiments as well) on the one hand, and ethical considerations on the other, were the reasons why we decided against sacrificing animals as a sham group without laparotomy.
In conclusion, our study demonstrates for the first time that S 100 B is released into serum by experimental hemorrhagic shock in rats, even in the absence of concomitant traumatic brain injury. In addition, S 100 B increase is associated with the severity of hemorrhagic shock. Further research will be necessary to trace this S 100 B increase to its source. It should also be kept in mind that in hemorrhagic shock with concomitant traumatic brain injury caused by direct impact to the head, brain-derived S 100 B may additionally contribute to peripheral serum S 100 B after trauma. These data are important because they provide the experimental basis for further evaluating the significance of S 100 B as a diagnostic parameter for the severity of hemorrhage in severely injured patients
The authors thank Mohammad Jafarmadar, Eva Toegel, and Christine Kober for technical assistance, and Ilse Jung for statistical evaluation.
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