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J Appl Physiol 103: 1021-1029, 2007. First published June 7, 2007; doi:10.1152/japplphysiol.01315.2006
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Severe hemodilutional anemia increases cerebral tissue injury following acute neurotrauma

Gregory M. T. Hare,1,2 C. David Mazer,1,2 James S. Hutchison,3,4 Anya T. McLaren,1 Elaine Liu,1 Alipasha Rassouli,1 Jinglu Ai,1 Rachel E. Shaye,3 Julia A. Lockwood,3 Cynthia E. Hawkins,5 Nancy Sikich,1 Kevin To,1 and Andrew J. Baker1

1Department of Anesthesia, Cara Phelan Centre for Trauma Research, Keenan Research Centre in the Li Ka Shing Knowledge Institute, University of Toronto, St. Michael's Hospital, Toronto; 2Department of Physiology, University of Toronto, Toronto; 3Departments of Critical Care Medicine and Paediatrics and the Neuroscience and Mental Health Program, The Hospital for Sick Children, Toronto; 4Interdepartmental Division of Critical Care, Faculty of Medicine, University of Toronto, Toronto; and 5Division of Neuropathology, Department of Laboratory Medicine, The Hospital for Sick Children, Toronto, Ontario, Canada

Submitted 20 November 2006 ; accepted in final form 4 June 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Anemia may worsen neurological outcomes following traumatic brain injury (TBI) by undefined mechanisms. We hypothesized that hemodilutional anemia accentuates hypoxic cerebral injury following TBI. Anesthetized rats underwent unilateral TBI or sham injury (n ≥ 7). Target hemoglobin concentrations between 50 and 70 g/l were achieved by exchanging 40–50% of the blood volume (1:1) with pentastarch. The effect of TBI, anemia, and TBI-anemia was assessed by measuring brain tissue oxygen tension (PbrO2), regional cerebral blood flow (rCBF), jugular venous oxygen saturation (SjvO2), cerebral contusion area, and nuclear staining for programmed cell death. Baseline postinjury PbrO2 values in the TBI and TBI-anemia groups (9.3 ± 1.3 and 11.3 ± 4.1 Torr, respectively) were lower than the uninjured controls (18.2 ± 5.2 Torr, P < 0.05 for both). Hemodilution caused a further reduction in PbrO2 in the TBI-anemia group relative to the TBI group without anemia (7.8 ± 2.7 vs. 14.8 ± 3.9 Torr, P < 0.05). The rCBF remained stable after TBI and increased comparably after hemodilution in both anemia and TBI-anemia groups. The SjvO2 was elevated after TBI (87.4 ± 8.9%, P < 0.05) and increased further following hemodilution (95.0 ± 1.6%, P < 0.05). Cerebral contusion area and nuclear counts for programmed cell death were increased following TBI-anemia (4.1 ± 3.0 mm2 and 686 ± 192, respectively) relative to TBI alone (1.3 ± 0.3 mm2 and 404 ± 133, respectively, P < 0.05 for both). Hemodilutional anemia reduced cerebral PbrO2 and oxygen extraction and increased cell death following TBI. These results support our hypothesis that acute anemia accentuated hypoxic cerebral injury after neurotrauma.

hemodilution; brain tissue oxygen tension

TRAUMATIC BRAIN INJURY (TBI) is the leading cause of death and severe disability in young adults and children (13, 29). Hypoxia and hypotension both contribute to secondary cerebral injury following TBI (7). However, the effect of acute anemia has not been established. Direct measurements of cerebral tissue oxygen tension have demonstrated that prolonged episodes of cerebral tissue hypoxia are associated with increased mortality (48, 50). Furthermore, systemic hypoxia has been demonstrated to increase the degree of secondary cerebral injury following neurotrauma in experimental and clinical studies (2, 7, 8, 23). These outcomes demonstrate that the brain is at risk of secondary injury following primary cerebral trauma. Hypoxia may augment cerebral injury following neurotrauma by promoting apoptosis (36).

Although the theory underlying regional cerebral oxygen supply and demand has been advanced for decades, the impact of normovolemic, normotensive, hemodilutional anemia on acute neurotrauma remains undefined. Decreased viscosity may optimize blood rheology during acute hemodilution. However, the associated reduction in blood oxygen content may limit regional cerebral oxygen delivery (46, 49). This issue is important to neurotrauma patients for three reasons: first, resuscitation with blood-free solutions is common following cerebral trauma (5); second, transfusion practices in critical care medicine have evolved such that physicians are targeting progressively lower transfusion thresholds (18); and third, hypoxic-ischemic insult is associated with worsened mortality and impaired functional outcomes following TBI (3). These concerns are underscored by a recent clinical analysis that suggests that patients with neurotrauma may have better outcomes if the hemoglobin concentration was maintained at a higher target with more blood transfusions (34). We hypothesize that acute hemodilution will reduce cerebral tissue oxygen tension and increase cerebral injury following acute neurotrauma. Target hemoglobin concentrations between 50 and 70 g/l were selected to test this hypothesis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Effect of TBI and Anemia on Brain Tissue Oxygen Tension and Regional Cerebral Blood Flow

Animal model.   All animal protocols were approved by the Animal Care and Use Committee at St. Michael's Hospital in accordance with the requirements of the Canadian Council on Animal Care. Male Sprague-Dawley rats (350–400 g, 11–12 wk old, Charles River, St. Constant, Quebec, Canada) were assigned to one of three groups: 1) TBI only (TBI, n = 7); 2) hemodilutional anemia (anemia, n = 8), or 3) TBI and anemia (TBI-anemia, n = 10). Animals were initially anesthetized with ketamine-xylazine (100 and 7.5 mg/kg ip, Parke-Davis/Bayer, Toronto, Ontario, Canada), and anesthesia was maintained with 2–3% isoflurane in 50% oxygen (Abbott, St. Laurent, Quebec, Canada). Following tracheostomy, rats were ventilated with a pressure-controlled ventilator (Kent Scientific, Litchfield, CT) to achieve normocapnia as determined by blood gas analysis (Radiometer ALB 500, London Scientific, London, Ontario, Canada). The right jugular vein (polyethylene 90), tail vein, and the tail artery (polyethylene 50) were cannulated to measure mean arterial blood pressure (MAP) and central venous pressure (CVP) and to perform hemodilution. Co-oximetry (Radiometer, OSM 3) and blood gas analysis were performed on arterial and venous blood. Animals were then placed in a stereotaxic frame (ADI Instruments, Harvard Apparatus, St. Laurent, Quebec, Canada), and their scalps were incised sagittally.

Fluid percussion injury.   A unilateral 5-mm-diameter burr hole was trephined over the left hemisphere at the level of bregma 0.0 to 5.0, 2 mm lateral to the sagittal sinus, to expose the intact dura. The pressure-conducting tube of the fluid percussion injury (FPI) device was then connected to the skull using acrylic cement. This provided a direct connection between the column of saline within the percussion device and the exposed dura. A calibrated 2-atmosphere (atm) impact was then performed as previously described (1). This degree of injury was utilized because it has been previously demonstrated to cause a unilateral region of brain injury (TBI) without causing evidence of histological damage within the contralateral cerebral hemisphere. The contralateral "uninjured" cerebral hemisphere could then serve as an internal control for experimental outcomes measured in the injured hemisphere. The 2-atm impact also correlates to clinical levels of injury associated with multiple trauma and systemic hemorrhage. Immediately postinjury, the cylinder was removed and a second burr hole was trephined over the right contralateral hemisphere at the corresponding position. The anemia group (sham TBI) was similarly attached to the FPI device, but no TBI was induced.

Subsequently, bilateral calibrated polarographic oxygen-sensing microelectrodes were inserted into the caudate nucleus using stereotaxic coordinates, as previously described (LICOX GMS, Harvard Apparatus) (15–17). Probes were inserted into the region of caudate nucleus approximately 4–6 mm past the dura, because this area is a large homogeneous region of gray matter with a high metabolic rate (45) that does not demonstrate evidence of cellular necrosis following TBI (2 atm). Placement of the probes was confirmed by brain dissection at the end of experimental procedures as previously validated (16, 17). The sensitivity of each probe was assessed by demonstrating its responsiveness to induced changes in partial pressure of arterial oxygen levels within the expected range of brain tissue oxygen tension (PbrO2) (11, 19, 31, 39). Bilateral temperature probes were placed at the same coordinates as the oximetry probes. Bilateral laser-Doppler flow probes (Oxyflo, Oxford Optronix, Oxford, UK) were positioned over the dura avoiding any visible large dural vessels. Measurements of regional cerebral blood flow (rCBF) were performed using probes at the surface of the cerebral cortex, remote from the oxygen electrode, to minimize local tissue trauma that might alter caudate tissue oxygen tension measurements. A period of 1 h was utilized to establish a steady baseline while a heating pad and heating lamp were used to maintain the brain temperature near 37°C. PbrO2, temperature, CBF, MAP, CVP, and heart rate (HR) were recorded digitally (DASYLab 5.6, Kent Scientific).

Hemodilutional anemia.   After a stable baseline was established, physiological parameters were measured for a total of 60 min. Rats were assigned to one of three groups as outlined above: 1) TBI alone (sham hemodilution, n = 7); 2) anemia alone (sham TBI, n = 8); and 3) TBI plus anemia (TBI-anemia, n = 10). A sham-sham control group was not performed as previously published experimental studies have demonstrated stable PbrO2 measurements near 18–20 Torr using these techniques (15, 17). In addition, the contralateral uninjured cerebral hemisphere served as an internal control to the effect of hemodilution in the TBI rats. In the two anemia groups, acute normovolemic hemodilutional anemia was induced over 10 min by simultaneous exchange of 30 ml/kg of arterial blood (50% estimated blood volume of 60 ml/kg), withdrawn from the tail artery, with an equivalent volume of pentastarch (Pentaspan, Bristol-Myers Squibb Canada, St. Laurent, Quebec, Canada) infused via the jugular vein. Volume exchange was performed over 10 min using a programmable "push-pull" pump (PHD 2000, Harvard Apparatus). Both MAP and CVP measurements were interrupted during the volume-exchange period. Following completion of volume exchange, all parameters were recorded for an additional 30 min before killing the animal by anesthetic overdose (ketamine 100 mg iv, Parke-Davis, Toronto, Ontario, Canada). In each group, arterial blood gas samples were taken at baseline and after hemodilution at 15-min intervals.

Effects of TBI and Anemia on Arterial and Jugular Venous Blood Gases

Three different groups of rats were utilized for these studies. Rats were anesthetized and ventilated as above and assigned to one of three groups: control (sham-sham, n = 6), anemia without TBI (anemia, n = 8), and TBI plus anemia (TBI-anemia, n = 6). Animals were prepared as described above with the exception that cannulation of the right jugular vein (polyethylene 90) was performed in a retrograde manner to allow for sampling of jugular venous blood. Venous blood samples were obtained before and after hemodilution by gravity-induced flow. The first 200 µl of each sample was discarded to minimize the effect of hemoconcentration secondary to venous occlusion between samples. Arterial and venous blood gas samples were taken simultaneously at similar time intervals as described above. The difference in arterial and venous oxygen content was calculated by subtracting corresponding venous and arterial values.

Effects of TBI and Anemia on Cerebral Contusion Area and TUNEL-Positive Cell Counts

Male Sprague-Dawley rats were anesthetized, intubated (14-gauge Angiocath), and ventilated before being assigned to one of three groups: control (sham-sham, n = 3), TBI alone (TBI, n = 7), and TBI plus anemia (TBI-anemia, n = 7). In this experiment, a less severe hemodilution protocol was utilized (40% estimated blood volume) by exchanging blood for pentastarch via the tail artery and vein to achieve a final target hemoglobin concentration near 70 g/l. This change allowed 100% survival to 5 days. In initial pilot experiments, hemodilution to a hemoglobin concentration of 50 g/l resulted in a survival rate near 70% with most deaths occurring within 12 h of surgery. Animals were then recovered and provided food and water ad libitum for 5 days. After 5 days, the rats were reanesthetized (2–3% isoflurane) and then perfused with 4% paraformaldehyde via a left ventricular catheter before whole brain extraction. After fixation, brain tissue was paraffin embedded. Whole mount coronal sections (5 µm) were prepared. Four coronal sections were obtained through the site of impact at the stereotaxic positions of bregma –2.3, –3.3 (anterior contusion), and bregma –5.3, –6.3 (posterior contusion). These sections were stained with cresyl violet and contusion areas were calculated for each section in a blinded manner, as previously described (36) [total magnification = 100x: eye piece (10x) x objective (10x), Leica DMLS Microscope, Wetzlar, Germany]. Microscopic images were captured and the area of contusion measured in the cortex of each rat ipsilateral to the site of injury using Image J image analysis software (version 1.37, National Institutes of Health, Bethesda, MD). The microscopists (JAL, RES) were blinded to the experimental group of the animals. The total cerebral contusion area was calculated by adding the contusion areas from each of the four histological sections for each rat. Two adjacent coronal sections (bregma –2.3 and –6.3) were utilized to assess the number of cells undergoing programmed cell death per coronal section in each rat as indicated by nuclear staining with terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling (TUNEL). TUNEL staining was performed utilizing a fluorescence in situ cell death detection kit as previously described (40) (Roche Applied Science, catalog no. 1684795, Laval, Quebec, Canada). TUNEL-positive immunofluorescent cells were counted in both injured and contralateral noninjured hemispheres of each coronal section at total magnification of 100x [eye piece (10x) x objective (10x), Olympus Provis Microscope]. Each cerebral hemisphere was divided into three sections, including 1) the contusion site, 2) the subcontusion cerebral cortex, and 3) the basal structures. Digital images were captured, and total TUNEL-positive cells were counted by blinded observers (ATM, RES). Patterns of TUNEL staining were compared, and the total number of TUNEL-positive nuclei were counted.

Statistical Analysis

Data are presented as means ± SD. Normal distribution of data was confirmed before statistical analysis was performed utilizing the SPSS software (SPSS, version 11.5.0). Physiological measurements (HR, MAP, CVP, PbrO2, and rCBF) were analyzed with a repeated-measures factorial ANOVA. The main effects of group, time, and hemisphere were assessed, and any interaction effects were defined. For cerebral tissue oxygen tension data, the baseline measurements for each cerebral hemisphere were held as covariates. Post hoc comparisons with Tukey's test were performed if statistical differences were determined by ANOVA. Comparison of MAP, PbrO2, and rCBF between groups was performed at 10 min (baseline) and after hemodilution at 40, 60, and 70 min. The total cerebral contusion area and the total number of TUNEL-positive nuclei were assessed as the primary outcomes of tissue injury using a Student's t-test. A Bonferonni adjustment was performed when appropriate. Statistical significance was assigned at the level of P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Effect of TBI and Anemia on PbrO2 and rCBF

With the exception of the PbrO2 measurements, there were no differences in any arterial blood gas, co-oximetry, or physiological parameter between the three groups at baseline (Table 1, Figs.1–4.) After hemodilution, there was a small increase in the MAP and CVP, relative to baseline, immediately following hemodilution, in both the anemia and TBI-anemia groups (Fig. 2, P < 0.05). However, there were no differences in MAP or CVP between any of the experimental groups at any time point (Fig. 2). Following hemodilution, the hemoglobin concentration in the anemia and TBI-anemia groups decreased comparably to minimum values of 56 ± 15 and 52 ± 13 g/l, respectively (Table 1, P < 0.05). The blood oxygen content was also reduced to a similar level in both groups.


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  		Table 1. Effect of hemodilution on co-oximetry and arterial blood gas measurements following TBI and anemia

 

Figure 1
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  		Fig. 1. Low brain oxygen tension was measured following traumatic brain injury (TBI). A: Mean arterial pressure remained stable near 80 mmHg. B: brain oxygen tension was measured using an oxygen-sensing microelectrode in the caudate nucleus. TBI resulted in a low brain oxygen tension, which increased slowly over time (*P < 0.05 vs. baseline). C: stable regional cerebral blood flow measured using laser-Doppler flowmetry in the cerebral cortex. Regional cerebral blood flow values are relative to a baseline value of 1.0.

 

Figure 4
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  		Fig. 4. Brain oxygen tension was reduced by TBI and further reduced by hemodilution. Three groups were compared: TBI, anemia alone, and TBI-anemia. A: mean arterial pressure remained near 80 mmHg with no differences between the 3 groups. B: brain tissue oxygen tension decreased following TBI at baseline. Following hemodilution, the brain oxygen tension decreased further in the TBI-anemia group relative to both TBI and anemia-only groups at 60 and 70 min. C: cerebral blood flow remained stable following TBI but was increased following hemodilution in both the TBI-anemia and anemia-alone groups. Regional cerebral blood flow values are relative to a baseline of 1.0. #P < 0.05, anemia vs. TBI and TBI-anemia groups; ##P < 0.05, anemia vs. TBI-anemia; $P < 0.05, TBI vs. TBI-anemia group. +P < 0.05, TBI vs. anemia and TBI-anemia groups.

 

Figure 2
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  		Fig. 2. Hemodynamics remained stable following normovolemic hemodilution. Hemodynamics were measured continuously before and after normovolemic hemodilutional anemia was induced over 10 min (black bars) by simultaneous exchange of 30 ml/kg of arterial blood with an equivalent volume of pentastarch. The effects of anemia and TBI-anemia were assessed by measuring heart rate (A), mean arterial pressure (B), and central venous pressure (C). Transient increases in the mean arterial and central venous pressures were observed in both groups following hemodilution (*P < 0.05 vs. baseline).

 
PbrO2 values were significantly reduced following injury in the TBI and TBI-anemia groups (9.3 ± 1.3 and 11.3 ± 4.1 Torr) relative to the anemia group (18.2 ± 5.2 Torr, Figs. 1, 3, and 4, P < 0.05 for both). Following TBI without hemodilution, the PbrO2 recovered slowly to a maximum value of 14.8 ± 3.9 Torr at 70 min (Fig. 1, P < 0.05), while the MAP and rCBF remained stable. In the TBI-anemia group, hemodilution resulted in a further reduction in PbrO2 to 7.8 ± 2.7 Torr by 70 min postinjury in the injured cerebral hemisphere (Fig. 3, P < 0.05), and this was significantly lower than the TBI group with no anemia (Fig. 4, P < 0.05). In the anemia group, the PbrO2 values remained relatively stable in both cerebral hemispheres following hemodilution (Fig. 3).


Figure 3
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  		Fig. 3. Decreased brain tissue oxygen tension with maintained cerebral blood flow in the injured hemisphere following hemodilution. A: baseline brain tissue oxygen tension was decreased in the injured cerebral hemisphere following TBI. Hemodilutional anemia (black bar) led to a further decrease in tissue oxygen tension compared with baseline measurements (prehemodilution) and the anemia alone group. B: brain tissue oxygen tension were not decreased in the contralateral hemisphere following anemia or TBI-anemia. C and D: cerebral blood flow increased in both the injured and contralateral cerebral hemispheres of the anemia and TBI-anemia rats. Regional cerebral blood flow values are relative to a baseline of 1.0. *P < 0.05 vs. baseline or prehemodilution. #P < 0.05 between anemia and TBI-anemia groups (injured hemisphere).

 
rCBF remained stable following TBI alone (Fig. 1). Following hemodilution, the rCBF increased significantly, relative to baseline, in both cerebral hemispheres, in both anemia and TBI-anemia groups (Figs. 3 and 4, P < 0.05 relative to baseline). However, there were no differences in the CBF response between anemia and TBI-anemia groups for either cerebral hemisphere (Fig. 4).

Effects of TBI and Anemia on Arterial and Jugular Venous Blood Gases

In the control group (no TBI or hemodilution), the arterial and venous co-oximetry and arterial blood gas measurements remained stable without any significant changes over time (data not shown). Hemodilution resulted in comparable reductions in arterial and venous hemoglobin concentrations and oxygen contents in both the anemia and TBI-anemia groups, relative to baseline values (Table 2, P < 0.05 for both). Arterial oxygen saturations remained between 97% and 100% in both groups. The baseline jugular venous oxygen saturation (SjvO2) was increased in the TBI-anemia group (87.4 ± 8.9%, Table 2, Fig. 5, P < 0.05) relative to the anemia group without TBI (69.1 ± 6.4%). Following hemodilution there was a small transient rise in the SjvO2 in the anemia group to 77.9 ± 2.6% and a larger increase in the TBI-anemia group (95.0 ± 1.6%) (Table 2, Fig. 5, P < 0.05, relative to baseline and between groups). The SjvO2 remained increased for up to 45 min in the TBI-anemia group relative to the anemia group following hemodilution (Table 2, Fig. 5, P < 0.05 between groups) but was not different from the anemia group at 60 min. The arterial-venous oxygen content difference was significantly reduced in the TBI-anemia group relative to the anemia group (Fig. 5, P < 0.05).


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  		Table 2. Effect of hemodilution and TBI on arterial and venous co-oximetry and blood gas analysis

 

Figure 5
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  		Fig. 5. Jugular venous saturation was abnormally high following hemodilution in rats with TBI. A: jugular venous oxygen saturation was higher in the TBI-anemia rats at baseline following TBI, and further increased following hemodilution, before returning to control values by 60 min. B: these changes were reflected in lower differences between arterial and jugular venous oxygen content observed in TBI-anemia rats following hemodilution, suggesting that cerebral oxygen extraction was reduced following TBI. *P < 0.05 vs. baseline, #P < 0.05 between groups.

 
Effects of TBI and Anemia on Cerebral Contusion Area and TUNEL-Positive Cell Counts

Control (no TBI or hemodilution) rats did not exhibit any evidence of cerebral contusion (cresyl violet) and had less than 25 TUNEL-positive cells per cerebral hemisphere (data not shown). There was a significant reduction in the hemoglobin concentration in the TBI-anemia group relative to the TBI group (68 ± 5 vs. 140 ± 3 g/l, respectively, P < 0.05). In the TBI and TBI-anemia groups, two areas of cerebral contusion were identified within the cerebral cortex. One area was located immediately superficial to the impact site (a), and the second area was located deeper within the cerebral cortex (b) (Fig. 6). The contusion area was larger in the posterior sections at bregma –5.3 and –6.3 relative to the anterior sections at bregma –2.3 and –3.3 in both groups (Figs. 6 and 7). After 5 days recovery, the total contusion area in the TBI-anemia group was significantly larger than that measured in the TBI group that underwent sham hemodilution (4.1 ± 3.0 vs. 1.3 ± 0.3 mm2, Fig. 7, P < 0.05). There was also a corresponding increase in the number of TUNEL-positive cells in the TBI-anemia group relative to TBI alone [686 ± 192 vs. 404 ± 133 nuclei per coronal section, respectively (Fig. 7, P < 0.05)]. The majority of TUNEL staining occurred in the cerebral cortex at the site of contusion or immediately inferior in the subcontusion region. Less staining was observed in the basal structures (Fig. 7). Very little TUNEL staining was observed in the contralateral uninjured cerebral hemisphere.


Figure 6
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  		Fig. 6. Cerebral contusion area and cell death (TUNEL) were increased by normovolemic hemodilution following TBI. A: diagram of coronal section of rat brain showing where the contusion area and TUNEL-positive cells were quantified. B: diagram of sagittal section of rat brain showing where coronal sections were taken. C and D: representative coronal sections of rat brain stained with cresyl violet and TUNEL, contrasting contusion area between TBI and TBI-Anemia groups. The 2 areas of contusion are labeled as superficial (a) and deep (b) to the site of impact. The cerebral contusion was larger in the posterior sections (e.g., bregma –6.3) compared with the anterior sections (e.g., bregma –3.3). Representative TUNEL-stained sections are shown at bregma –6.3. The white and black scale bars represent 100 µm in all cases.

 

Figure 7
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  		Fig. 7. Cerebral contusion area and TUNEL-positive cells counts were significantly increased by hemodilution following TBI. A: total contusion area and TUNEL counts were increased at 5 days in the TBI-anemia group relative to TBI only. There was no contusion seen on cresyl violet staining in the contralateral hemisphere in injured rats, or in sham rats (data not shown). B: examples of TUNEL staining. C: comparison of the number of TUNEL-positive nuclei in the TBI and TBI-anemia groups in the injured and contralateral hemispheres. TUNEL staining was minimal in the contralateral hemisphere (#P < 0.05 between groups).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
We have demonstrated that acute anemia induced by normovolemic hemodilution led to cerebral hypoxia and accentuated cerebral injury in an experimental model of neurotrauma. Following unilateral traumatic brain injury, acute normovolemic hemodilution reduced regional cerebral tissue oxygen tension, while SjvO2 and rCBF were elevated, suggesting that impairment of oxygen extraction had occurred. Acute normovolemic hemodilution also increased cerebral cortical tissue contusion area and cell death following TBI as measured in brain sections stained with cresyl violet and TUNEL. We also demonstrated that cerebral oxygen tension is maintained in the normal range and that no cerebral injury occurred following hemodilution without TBI. These data suggest that the brain is selectively vulnerable to anemia following neurotrauma.

The relevance of our experimental results is supported by a subanalysis of a clinical trial that suggests that patients suffering from acute neurotrauma may benefit from a higher transfusion threshold (18, 34). In addition, the "Lund therapy" management protocol, which contains higher transfusion thresholds, may lead to improved outcomes following acute neurotrauma (12). A recent clinical study in patients following cardiopulmonary bypass has demonstrated that relatively small changes in cerebral tissue oxygen tension may have an important impact on patient morbidity (38). Acute hemodilution is associated with increased neurological injury and higher mortality following cardiopulmonary bypass in animal models and in both adult and pediatric patients (14, 20, 25). The results of these studies, in combination with other studies showing that cerebral tissue oxygen tension is maintained during hemodilution in the absence of brain injury (11, 19, 31, 39, 44, 49), support our conclusion that the brain is selectively vulnerable to normovolemic hemodilution following TBI.

Reduced tissue oxygen tension may have a significant impact on neurological outcomes since relatively mild levels of systemic hypoxia have been shown to augment secondary cerebral injury following neurotrauma (2, 7, 8, 23, 36). Possible mechanisms of this selective vulnerability to hypoxia include impaired CBF responses and depletion of high-energy phosphates following TBI (10, 24). Additional experimental studies utilizing the current model have provided evidence that hemodilution triggers an increased expression of hypoxic molecules within the cerebral cortex, including hypoxia inducible factor-1{alpha}, neuronal nitric oxide synthase, and vascular endothelial growth factor and chemokine receptors (17, 35). These molecules may contribute to adaptive regulatory mechanisms following hemodilution. Alternately, they may contribute to secondary neuronal injury following TBI and hemodilution by mechanisms that include inflammation, oxidative stress, and apoptosis (26, 27, 32, 36, 41).

The increase in CBF associated with hemodilution is a well-characterized phenomenon caused by passive changes in rheology and active cerebral vasodilation (4, 22, 46). This CBF response may optimize cerebral oxygen delivery during anemia, thereby defending against cerebral tissue hypoxia (10). This response is observed in most areas of gray matter, including the caudate nucleus (42). CBF is initially reduced and autoregulation is impaired following TBI (10). In our study, the increase in CBF in response to hemodilution was relatively preserved in the injured and contralateral cerebral cortexes. However, the associated increase in SjvO2 suggests that impairment of microcirculatory function may have led to increased shunting through larger conductance vessels and reduced cerebral oxygen extraction.

Experimental studies suggest that the cerebral metabolic rate for oxygen (CMRO2) is immediately increased following TBI (6, 30). However, this initial state of hypermetabolism rapidly evolves into a more sustained state of hypometabolism (10, 30), possibly secondary to impaired mitochondrial function (9, 21). Early cerebral hypermetabolism cannot fully explain the reduction in cerebral tissue oxygen tension observed following TBI because such hypermetabolism would be expected to reduce the SjvO2. The existing literature suggests that there is a decrease in oxygen consumption after TBI. Nonetheless, reduction in blood oxygen-carrying capacity with severe hemodilution can lead to inadequate regional cerebral tissue oxygen delivery and increased tissue damage. After 60 min, the SjvO2 returned to baseline, possibly because of compensatory mechanisms that restored oxygen extraction (49).

There are some limitations to this study. Only one level of trauma was evaluated as part of the experimental design. The level of TBI was chosen to approximate the level of clinical injury that is often associated with systemic hemorrhage. Although target hemoglobin values near 50 and 70 g/l were used, future studies will be required to establish the hemoglobin threshold at which secondary injury occurs following TBI and anemia. We did not measure intracranial pressure, which is important in the assessment of cerebral perfusion. We did measure rCBF, however, which may be a better index of cerebral perfusion. We used laser-Doppler flowmetry to measure red blood cell flux since it correlates strongly with CBF in previous studies in animal models (28, 33). Tissue oxygen tension and laser-Doppler flow probes were located in different regions of the brain to minimize the adverse effect of local tissue damage at the site of the oxygen electrode on rCBF measurements. Furthermore, comparable changes in cortical and caudate tissue blood flow have been demonstrated following hemodilution in another experimental study (43). Pentastarch was chosen for hemodilution because volume exchange with synthetic starch colloids has been previously utilized to maintain adequate preload and prevent any significant reduction in blood pressure (37, 42, 46, 47). Maintenance of blood pressure was important because of the identified negative impact of systemic hypotension on cerebral tissue oxygen tension and neurological outcomes following TBI (19, 31, 37).

In summary, acute normovolemic hemodilutional anemia reduced cerebral tissue oxygen tension in the contused brain. This was associated with an increase in SjvO2, during a period of increased CBF, suggesting that reduced cerebral oxygen extraction within the cerebral microcirculation likely contributed to the observed reduction in cerebral tissue oxygen tension. The increase in cerebral tissue contusion area and TUNEL-positive cell counts suggest that anemia leads to secondary cerebral injury following TBI. The evolving practice of restricting blood transfusion in critically ill patients might not be appropriate for patients with TBI. Optimal transfusion thresholds for patients with TBI may be different from other critically ill patients. Further research is required to determine if maintaining a higher hematocrit in patients suffering from TBI would improve clinical outcomes.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
 REFERENCES
 
This research was supported by the Canadian Anesthesiologists' Society, Physicians' Services Incorporated Foundation, Anemia Institute for Research and Education, Bayer-Canadian Blood Services-Hema Quebec-Canadian Institutes of Health Research Partnership Fund, the Ontario Neurotrauma Foundation, and the St. Michael's Hospital Department of Anesthesia. G. M. T. Hare is the recipient of the Bristol-Myers Squibb-Canadian Anesthesiologists' Society Career Scientist Award in Anesthesia and Perioperative Medicine.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
We acknowledge Dr. Brian Kavanagh for critical appraisal of the manuscript and Eugene Park, Rong Qu, Julie Yu, and Jordan Worrall for technical assistance.

A portion of this study was presented at the National Neurotrauma Society, Biloxi MS, November 6–7, 2003, and has appeared in abstract form (17a).


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. M. T. Hare, Dept. of Anesthesia, Univ. of Toronto, St. Michael's Hospital, 30 Bond St., Toronto, Ontario M5B 1W8, Canada (e-mail: hareg{at}smh.toronto.on.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 

  1. Baker AJ, Phan N, Moulton RJ, Fehlings MG, Yucel Y, Zhao M, Liu E, Tian GF. Attenuation of the electrophysiological function of the corpus callosum after fluid percussion injury in the rat. J Neurotrauma 19: 587–599, 2002.[CrossRef][ISI][Medline]
  2. Bauman RA, Widholm JJ, Petras JM, McBride K, Long JB. Secondary hypoxemia exacerbates the reduction of visual discrimination accuracy and neuronal cell density in the dorsal lateral geniculate nucleus resulting from fluid percussion injury. J Neurotrauma 17: 679–693, 2000.[CrossRef][ISI][Medline]
  3. Baxt WG, Moody P. The differential survival of trauma patients. J Trauma 27: 602–606, 1987.[ISI][Medline]
  4. Brown MM, Wade JP, Marshall J. Fundamental importance of arterial oxygen content in the regulation of cerebral blood flow in man. Brain 108: 81–93, 1985.[Abstract/Free Full Text]
  5. Bullock R, Chesnut RM, Clifton G, Ghajar J, Marion DW, Narayan RK, Newell DW, Pitts LH, Rosner MJ, Wilberger JW. Guidelines for the management of severe head injury. Brain Trauma Foundation Eur J Emerg Med 3: 109–127, 1996.
  6. Chen T, Qian YZ, Di X, Rice A, Zhu JP, Bullock R. Lactate/glucose dynamics after rat fluid percussion brain injury. J Neurotrauma 17: 135–142, 2000.[ISI][Medline]
  7. Chesnut RM, Marshall LF, Klauber MR, Blunt BA, Baldwin N, Eisenberg HM, Jane JA, Marmarou A, Foulkes MA. The role of secondary brain injury in determining outcome from severe head injury. J Trauma 34: 216–222, 1993.[ISI][Medline]
  8. Clark RS, Kochanek PM, Dixon CE, Chen M, Marion DW, Heineman S, DeKosky ST, Graham SH. Early neuropathologic effects of mild or moderate hypoxemia after controlled cortical impact injury in rats. J Neurotrauma 14: 179–189, 1997.[ISI][Medline]
  9. Clausen T, Zauner A, Levasseur JE, Rice AC, Bullock R. Induced mitochondrial failure in the feline brain: implications for understanding acute post-traumatic metabolic events. Brain Res 908: 35–48, 2001.[CrossRef][ISI][Medline]
  10. DeWitt DS, Prough DS. Traumatic cerebral vascular injury: the effects of concussive brain injury on the cerebral vasculature. J Neurotrauma 20: 795–825, 2003.[CrossRef][ISI][Medline]
  11. Dunn JF, Nwaigwe CI, Roche M. Measurement of arterial, venous, and interstitial PO2 during acute hypoxia in rat brain using a time-resolved luminescence-based oxygen sensor. Adv Exp Med Biol 471: 43–48, 1999.[ISI][Medline]
  12. Eker C, Asgeirsson B, Grande PO, Schalen W, Nordstrom CH. Improved outcome after severe head injury with a new therapy based on principles for brain volume regulation and preserved microcirculation. Crit Care Med 26: 1881–1886, 1998.[ISI][Medline]
  13. Finnie JW, Blumbergs PC. Traumatic brain injury. Vet Pathol 39: 679–689, 2002.[Abstract/Free Full Text]
  14. Habib RH, Zacharias A, Schwann TA, Riordan CJ, Durham SJ, Shah A. Adverse effects of low hematocrit during cardiopulmonary bypass in the adult: should current practice be changed? J Thorac Cardiovasc Surg 125: 1438–1450, 2003.[Abstract/Free Full Text]
  15. Hare GMT, Kavanagh BP, Mazer CD, Hum KM, Kim SY, Coackley C, Barr A, Baker AJ. Hypercapnia increases cerebral tissue oxygen tension in anesthetized rats. Can J Anaesth 50: 1061–1068, 2003.[Abstract/Free Full Text]
  16. Hare GMT, Hum KM, Kim SY, Barr A, Baker AJ, Mazer CD. Increased cerebral tissue oxygen tension after extensive hemodilution with a hemoglobin-based oxygen carrier. Anesth Analg 99: 528–35, 2004.[Abstract/Free Full Text]
  17. Hare GMT, Mazer CD, Mak W, Gorczynski RM, Hum KM, Kim SY, Wyard L, Barr A, Qu R, Baker AJ. Hemodilutional anemia is associated with increased cerebral neuronal nitric oxide synthase gene expression. J Appl Physiol 94: 2058–2067, 2003.[Abstract/Free Full Text]
  18. Hare GMT, Mazer CD, Rassouli AP, Tian GF, Qu R, To K, Sikich N, Baker AJ. Hemodilutional anemia accentuates anemia-induced cerebral hypoxia following traumatic brain injury. J Neurotrauma 20: 1134, 2003.
  19. Hebert PC, Wells G, Blajchman MA, Marshall J, Martin C, Pagliarello G, Tweeddale M, Schweitzer I, Yetisir E. A multicenter, randomized, controlled clinical trial of transfusion requirements in critical care. Transfusion Requirements in Critical Care Investigators, Canadian Critical Care Trials Group. N Engl J Med 340: 409–417, 1999.[Abstract/Free Full Text]
  20. Hemphill JC, Knudson MM, Derugin N, Morabito D, Manley GT. Carbon dioxide reactivity and pressure autoregulation of brain tissue oxygen. Neurosurgery 48: 377–383, 2001.[CrossRef][ISI][Medline]
  21. Homi HM, Yang H, Pearlstein RD, Grocott HP. Hemodilution during cardiopulmonary bypass increases cerebral infarct volume after middle cerebral artery occlusion in rats. Anesth Analg 99: 974–981, 2004.[Abstract/Free Full Text]
  22. Hovda DA, Yoshino A, Kawamata T, Katayama Y, Becker DP. Diffuse prolonged depression of cerebral oxidative metabolism following concussive brain injury in the rat: a cytochrome oxidase histochemistry study. Brain Res 567: 1–10, 1991.[CrossRef][ISI][Medline]
  23. Hudak ML, Koehler RC, Rosenberg AA, Traystman RJ, Jones MD. Effect of hematocrit on cerebral blood flow. Am J Physiol Heart Circ Physiol 251: H63–H70, 1986.[Abstract/Free Full Text]
  24. Ishige N, Pitts LH, Hashimoto T, Nishimura MC, Bartkowski HM. Effect of hypoxia on traumatic brain injury in rats. 1. Changes in neurological function, electroencephalograms, and histopathology. Neurosurgery 20: 848–853, 1987.[ISI][Medline]
  25. Ishige N, Pitts LH, Pogliani L, Hashimoto T, Nishimura MC, Bartkowski HM, James TL. Effect of hypoxia on traumatic brain injury in rats. 2. Changes in high energy phosphate metabolism. Neurosurgery 20: 854–858, 1987.[ISI][Medline]
  26. Jonas RA, Wypij D, Roth SJ, Bellinger DC, Visconti KJ, du Plessis AJ, Goodkin H, Laussen PC, Farrell DM, Bartlett J, McGrath E, Rappaport LJ, Bacha EA, Forbess JM, del Nido PJ, Mayer JE Jr, Newburger JW. The influence of hemodilution on outcome after hypothermic cardiopulmonary bypass: results of a randomized trial in infants. J Thorac Cardiovasc Surg 126: 1765–1774, 2003.[Abstract/Free Full Text]
  27. Keane RW, Kraydieh S, Lotocki G, Alonso OF, Aldana P, Dietrich WD. Apoptotic and antiapoptotic mechanisms after traumatic brain injury. J Cereb Blood Flow Metab 21: 1189–1198, 2001.[ISI][Medline]
  28. Knoblach SM, Faden AI. Administration of either anti-intercellular adhesion molecule-1 or a nonspecific control antibody improves recovery after traumatic brain injury in the rat. J Neurotrauma 19: 1039–1050, 2002.[CrossRef][ISI][Medline]
  29. Kramer MS, Vinall PE, Katolik LI, Simeone FA. Comparison of cerebral blood flow measured by laser-Doppler flowmetry and hydrogen clearance in cats after cerebral insult and hypervolemic hemodilution. Neurosurgery 38: 355–361, 1996.[CrossRef][ISI][Medline]
  30. Kraus JF, McArthur DL. Epidemiologic aspects of brain injury. Neurol Clin 14: 435–450, 1996.[CrossRef][ISI][Medline]
  31. Levasseur JE, Alessandri B, Reinert M, Bullock R, Kontos HA. Fluid percussion injury transiently increases then decreases brain oxygen consumption in the rat. J Neurotrauma 17: 101–112, 2000.[ISI][Medline]
  32. Manley GT, Pitts LH, Morabito D, Doyle CA, Gibson J, Gimbel M, Hopf HW, Knudson MM. Brain tissue oxygenation during hemorrhagic shock, resuscitation, and alterations in ventilation. J Trauma 46: 261–267, 1999.[ISI][Medline]
  33. Marklund N, Clausen F, McIntosh TK, Hillered L. Free radical scavenger posttreatment improves functional and morphological outcome after fluid percussion injury in the rat. J Neurotrauma 18: 821–832, 2001.[CrossRef][ISI][Medline]
  34. Mazzoni MC, Warnke KC, Arfors KE, Skalak TC. Capillary hemodynamics in hemorrhagic shock and reperfusion: in vivo and model analysis. Am J Physiol Heart Circ Physiol 267: H1928–H1935, 1994.[Abstract/Free Full Text]
  35. McIntyre LA, Ferguson DA, Hutchison JS, Pagliarello G, Marshall JC, Yetisir E, Hare GMT, Hebert PC. Effect of a liberal versus restrictive transfusion strategy on mortality in patients with moderate to severe head injury. J Neuro Crit Care 5: 4–9, 2006.
  36. McLaren AT, Marsden PA, Mazer CD, Baker AJ, Stewart DJ, Tsui AK, Li X, Yucel Y, Robb M, Boyd SR, Liu E, Yu J, Hare GM. Increased expression of HIF-1{alpha}, nNOS, and VEGF in the cerebral cortex of anemic rats. Am J Physiol Regul Integr Comp Physiol 292: R403–R414, 2007.[Abstract/Free Full Text]
  37. Mikrogianakis A, Shaye RE, Griffen P, Kawesa S, Lockwood J, Gendron NH, Gaboury I, Merali Z, MacKenzie AE, Hutchison JS. Hypoxia alters the expression of inhibitors of apoptosis proteins after brain trauma in the mouse. J Neurotrauma 24: 338–53, 2007.[CrossRef][ISI][Medline]
  38. Morimoto Y, Mathru M, Martinez-Tica JF, Zornow MH. Effects of profound anemia on brain tissue oxygen tension, carbon dioxide tension, and pH in rabbits. J Neurosurg Anesthesiol 13: 33–39, 2001.[CrossRef][ISI][Medline]
  39. Murkin JM, Adams SJ, Novick RJ, Quantz M, Bainbridge D, Iglesias I, Cleland A, Schaefer B, Irwin B, Fox S. Monitoring brain oxygen saturation during coronary bypass surgery: a randomized, prospective study. Anesth Analg 104: 51–58, 2007.[Abstract/Free Full Text]
  40. Nwaigwe CI, Roche MA, Grinberg O, Dunn JF. Effect of hyperventilation on brain tissue oxygenation and cerebrovenous PO2 in rats. Brain Res 868: 150–156, 2000.[CrossRef][ISI][Medline]
  41. Park E, McKnight S, Ai J, Baker AJ. Purkinje cell vulnerability to mild and severe forebrain head trauma. J Neuropathol Exp Neurol 65: 226–234, 2006.[ISI][Medline]
  42. Raghavendra R, V, Dhodda VK, Song G, Bowen KK, Dempsey RJ. Traumatic brain injury-induced acute gene expression changes in rat cerebral cortex identified by GeneChip analysis. J Neurosci Res 71: 208–219, 2003.[CrossRef][ISI][Medline]
  43. Rebel A, Lenz C, Krieter H, Waschke KF, Van Ackern K, Kuschinsky W. Oxygen delivery at high blood viscosity and decreased arterial oxygen content to brains of conscious rats. Am J Physiol Heart Circ Physiol 280: H2591–H2597, 2001.[Abstract/Free Full Text]
  44. Rebel A, Ulatowski JA, Joung K, Bucci E, Traystman RJ, Koehler RC. Regional cerebral blood flow in cats with cross-linked hemoglobin transfusion during focal cerebral ischemia. Am J Physiol Heart Circ Physiol 282: H832–H841, 2002.[Abstract/Free Full Text]
  45. Shen H, Greene AS, Stein EA, Hudetz AG. Functional cerebral hyperemia is unaffected by isovolemic hemodilution. Anesthesiology 96: 142–147, 2002.[CrossRef][ISI][Medline]
  46. Sokoloff L, Reivich M, Kennedy C, Des Rosiers MH, Patlak CS, Pettigrew KD, Sakurada O, Shinohara M. The [14C]deoxyglucose method for the measurement of local cerebral glucose utilization: theory, procedure, and normal values in the conscious and anesthetized albino rat. J Neurochem 28: 897–916, 1977.[ISI][Medline]
  47. Todd MM, Wu B, Maktabi M, Hindman BJ, Warner DS. Cerebral blood flow and oxygen delivery during hypoxemia and hemodilution: role of arterial oxygen content. Am J Physiol Heart Circ Physiol 267: H2025–H2031, 1994.[Abstract/Free Full Text]
  48. Tsai AG. Influence of cell-free Hb on local tissue perfusion and oxygenation in acute anemia after isovolemic hemodilution. Transfusion 41: 1290–1298, 2001.[CrossRef][ISI][Medline]
  49. Valadka AB, Gopinath SP, Contant CF, Uzura M, Robertson CS. Relationship of brain tissue PO2 to outcome after severe head injury. Crit Care Med 26: 1576–1581, 1998.[CrossRef][ISI][Medline]
  50. van Bommel J, Trouwborst A, Schwarte L, Siegemund M, Ince C, Henny C. Intestinal and cerebral oxygenation during severe isovolemic hemodilution and subsequent hyperoxic ventilation in a pig model. Anesthesiology 97: 660–670, 2002.[CrossRef][ISI][Medline]
  51. van den Brink WA, van Santbrink H, Steyerberg EW, Avezaat CJ, Suazo JA, Hogesteeger C, Jansen WJ, Kloos LM, Vermeulen J, Maas AI. Brain oxygen tension in severe head injury. Neurosurgery 46: 868–876, 2000.[CrossRef][ISI][Medline]



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