Neutrophil Accumulation After Traumatic Brain Injury in

Rats: Comparison of Weight Drop and Controlled

Cortical Impact Models

ROBERT S.B. CLARK,1 JOANNE K. SCHIDING,2 SUSAN L. KACZOROWSKI,1

DONALD W. MARION,3 and PATRICK M. KOCHANEK1,2

ABSTRACT

Previous work in our laboratory and others using the weight drop (WD) model of traumatic
brain injury (TBI) has shown that neutrophils accumulate in brain tissue during the initial 24
h posttrauma as measured by myeloperoxidase (MPO) activity and immunohistochemistry.
This study compares the acute inflammatory response to TBI over time, as measured by MPO
activity, in the WD and controlled cortical impact (CCI) models. Anesthetized adult Sprague\x=req-\
Dawley rats were traumatized using WD (10-g weight dropped 5 cm) or CCI (4 m/sec, 2.5 mm
depth). At 2, 24, 48, or 168 h after trauma, rats (n = 4\p=n-\5/group at each time) were anesthetized
and killed, the brains were removed, and 6-mm coronal slices from traumatized and con-
tralateral hemispheres were assayed for MPO activity. Nontraumatized rats (n = 4) served as
controls. Three additional rats underwent a more severe CCI (3 mm depth) with MPO activity
assayed at 24 h. A separate group of rats (n = 6) was subjected to WD trauma and killed at 2
weeks after injury for analysis of lesion volume. MPO activity in the traumatized hemisphere
was demonstrated at 24 and 48 h in both the WD (0.3152 \m=+-\0.0472 and 0.3017 \m=+-\0.0228 U/g, re-

spectively, p < 0.05 vs controls) and CCI (0.1866 \m=+-\0.0225 and 0.1937 \m=+-\0.0772 U/g, respec-
tively, p < 0.05 vs controls) models. MPO activity was below the sensitivity of the assay in the
control, 2 h, and 168 h groups in both models. MPO activity at 24 h after CCI increased with
the depth of injury (0.1866 \m=+-\0.0225 vs 0.3011 \m=+-\0.0141 U/g, 2.5 vs 3.0 mm depth, respectively,
p < 0.05). Lesion volume at 2 weeks after WD trauma was 16.0 \m=+-\2.7 mm3,17.5% larger than
lesion volume after CCI (previously published data), but the difference between groups was
not statistically significant. These data indicate that (1) MPO activity after traumatic brain in-
jury is increased at 24 and 48 h and resolves by 168 h in both the WD and CCI models, and (2)
MPO activity in the CCI model increases with the severity of trauma.

INTRODUCTION
may play A key role in secondary brain injury after trauma, specifically contributing to al-
Inflammation
terations in cerebral blood flow, edema, intracranial hypertension, and ultimately neuronal death.

Departments of 'Pediatrics, 2Anesthesiology and Critical Care Medicine, and 'Neurological Surgery, Safar Center for
Resuscitation Research and the Brain Trauma Research Center, University of Pittsburgh, Pittsburgh, Pennsylvania.
Presented in part at the 11th Meeting of the Society of Neurotrauma, November 6-7,1993, Washington, DC.
Neutrophils, important in the inflammatory response outside of the central nervous system (CNS) (Issekutz,
1981 ; Täte and Repine, 1983), are thought to participate in the inflammatory response after brain injury as well
(Persson, 1976; Kochanek and Hallenbeck, 1992). The myeloperoxidase (MPO) assay is a sensitive and spe-
cific method of quantifying neutrophil accumulation (Bradley et al., 1982; Krawisz et al., 1984; Xu et al.,
1990). This assay was developed by Bradley et al. (1982) to measure neutrophil accumulation in inflammatory
skin lesions and was modified for use in brain by Barone et al. (1991) and in spinal cord injury by Xu et al.
(1990). Previous work in our laboratory (Biagas et al., 1992b) using the weight drop (WD) model of traumatic
brain injury (TBI) showed that neutrophils accumulate in brain tissue 24 h posttrauma as measured by MPO
activity. Horner et al. (1992) demonstrated that MPO activity was maximal at 24-72 h in a similar WD model
and verified that increases in MPO activity corresponded to neutrophil accumulation by immunohistochemical
methods.
Controlled cortical impact (CCI) is a contemporary model of TBI that reproduces important features of hu-
man head injury, including contusion and both local and diffuse axonal injury (Lighthall et al., 1990). The post-
traumatic histopathologic and behavioral effects of this model have been well characterized (Dixon et al.,
1991; Sutton et al., 1993). Published data describing the posttraumatic pafhophysiologic characteristics, such
as alterations in cerebral blood flow, edema, and intracranial hypertension in this model, are limited. Similarly,
no reports have described or quantified inflammatory cell infiltration after CCI.
This study examines the acute inflammatory response to cerebral trauma by evaluating the time course of
neutrophil accumulation in brain, as measured by MPO activity, and compares this response in two standard-
ized models of TBI, WD and CCI. The relation between ultimate lesion volume and MPO activity also is ex-
amined in the two models.

MATERIALS AND METHODS

Surgical Procedures
All studies were approved by the University of Pittsburgh Animal Care and Use Committee. All surgical
procedures were performed using aseptic technique. Forty-seven virus-free, mature male Sprague-Dawley rats
(older than 2 months, weighing 280-400 g) had free access to food and water until the time of the study. Rats
were assigned to one of three groups: (1) WD trauma (n 24), (2) CCI trauma (n 19), or (3) control (no
= =

trauma, n =
4).
Anesthesia was induced in a plasticjar with 4% isoflurane (Anaquest, Memphis, TN) in oxygen. The trachea
was intubated with a 14-gauge angiocatheter, and the lungs were mechanically ventilated with 1.1%—2.0%
isoflurane/66% N20/balance 02. A femoral arterial catheter was inserted for continuous monitoring of blood
pressure, arterial blood sampling, and administration of pancuronium bromide (0.1 mg/kg/h, Elkins-Sinn,
Cherry Hill, NJ). A rectal probe was inserted for continuous monitoring and maintenance of temperature at
37.0°C ± 0.5°C, with the aid of a heated water blanket. Bicillin (100,000 U, Upjohn, Kalamazoo, MI) and gen-
tamicin (10 mg/kg, Elkins-Sinn) were administered intramuscularly.
In preparation for trauma, the head was fixed in a stereotactic device (David Kopf, Tujunga, CA). After re-
traction of the scalp, a craniotomy was made over the parietal cortex [right for WD, left for CCI, as previously
described (Schoettle et al., 1990; Palmer et al., 1993)] with a dental drill, using the coronal and interparietal su-
tures as margins. The intact dura and bone flap were left in place, and the rats were allowed to equilibrate un-
der anesthesia (1.1% isoflurane/66% N20/balance 02) for 30 min. Fifteen minutes before trauma, an arterial
blood sample (0.5 mL) was obtained to verify that arterial blood gas tensions and hematocrit were within nor-
mal limits.
TBI using the WD method was performed in 24 rats as previously described (Schoettle et al., 1990). After
removal of the bone flap, injury was produced by dropping a 10-g brass rod (3.0 mm diameter, 15.2 cm length)
from a distance of 5 cm through a glass guide tube onto the exposed dura over the right parietal cortex.
TBI using the CCI method was performed in 19 rats as previously described (Palmer et al., 1993). After re-
moval of the bone flap, injury to the left parietal cortex was produced using the CCI device (Lighthall et al.,
1990; Dixon et al., 1991). This device consists of a 5-mm metal impactor tip that is pneumatically driven at a
predetermined velocity, depth, and duration of brain deformation. For all studies, velocity was 4.0 ± 0.2 m/sec,
and duration of deformation was 50 msec. Depth of penetration was 2.5 mm for moderate injury and 3.0 mm
for severe injury.
Following trauma, the bone flaps were replaced and sealed with dental cement (Koldmount, Vernon
Benshoff Co, Albany, NY), and the scalp incisions were closed. Femoral catheters and temperature probes
were removed, and the rats were weaned from mechanical ventilation and extubated within 1 h. Animals were
observed in 100% oxygen for 30 min, then returned to their cages and allowed free access to food and water.

MPO Activity Assay

At either 2, 24, 48, or168 h after trauma, rats (n 4-5 per group) were reanesthetized as previously de-
=

scribed. Through midline thoracotomy, the aorta was clamped at the midthoracic level. To eliminate in-
a
travascular blood, the head and upper torso were perfused transcardially with 200 mL of normal saline through
the left ventricle. Normal rats were anesthetized and transcardially perfused in a similar fashion. Brains were
removed, and 6-mm coronal sections centered on the traumatized area were obtained. Traumatized and con-
tralateral hemispheres were separated and weighed. The MPO assay was performed on the 6-mm coronal sec-
tions from both the traumatized and contralateral hemispheres.
Each brain tissue sample was homogenized in 8 mL of 50 mM potassium phosphate buffer, pH 6.0 (Sigma,
St. Louis, MO) and was centrifuged at 2500g for 10 min. The supernatant was discarded, and the pellet was re-
suspended in 8 mL of buffer. Samples were washed in potassium phosphate buffer and centrifuged twice to re-
move inhibitors of MPO activity (Barone et al., 1991).
MPO activity was measured in brain tissue homogenates from traumatized and normal rats. Brain tissue pel-
lets were resuspended in 2.5 mL of hexadecyltrimethylammonium bromide (HTAB, 0.5% HTAB in 50 mM
potassium phosphate buffer, pH 6.0, Sigma). HTAB is a detergent that liberates the MPO enzyme from leuko-
cyte granules. Three rapid freeze-fhaw cycles were performed to further disrupt leukocyte granules. Samples
were centrifuged for 10 min at 2500g for a final time. Supernatant (100 pL) was added to 2.9 mL of 50 mM

potassium phosphate buffer with o-diasinidine hydrochloride (0.167 mg/mL, Sigma) and H202 (0.0005%,
Sigma). Change in absorbance at 460 nm was assayed spectrophotometrically (Beckman, Somerset, NJ) for 2
min in triplicate, with MPO activity determined from the mean of the three readings. One unit of MPO activity
was defined as the degradation of 1 pmol of H202/min (Barmen, 1969). Results were expressed as units of
MPO activity per gram of tissue.
A standard curve for the MPO assay was determined using peritoneal neutrophils obtained from rats (n 4)
after the intraperitoneal injection of 300 mg of casein (Schoettle et al., 1990). Isolated neutrophils (50 x 106) in
-

50 mM potassium phosphate buffer were added to brain homogenate and then prepared as described previ-
ously. MPO activity was assayed in 0.1 mL samples from serial dilutions (5 x 103—1 x 107 neutrophils/mL) of
the neutrophil-brain homogenate suspension. A standard curve was constructed and was linear in the range of
2 x 103—1 x 105 neutrophils/sample, as described by Xu et al. (1990).

Lesion Volume
Fourteen days after WD trauma, 6 rats were anesthetized with isoflurane and perfused with 4%
paraformaldehyde in phosphate-buffered saline (10 mM sodium phosphate, 154 mM NaCl, pH 7.4). Using a
microtome, serial 40-pm frozen sections were cut in the coronal plane at 1-mm distances from the occiput and
mounted on glass slides. Three to four sections were obtained at each distance and stained with hematoxylin
and eosin. The area of the lesion was determined in each coronal slice using an image analysis system (MCID,
Imaging Research, St. Catherines, Ontario, Canada). The area at each distance was determined by averaging
the lesion areas for each slice prepared at that distance. The areas at each distance were summed, and lesion
volume was calculated.

Statistical Analysis
All data are presented as mean ± SEM. Within-group comparisons of MPO activity (control, 2, 24,48, and
168 h after WD and CCI trauma) were made using one-way ANO VA and the Student-Newman-Keuls test.
Comparison of MPO activity as a function of severity of injury in the CCI model was also made using one-way
ANOVA and the Student-Newman-Keuls test. Intermodel comparisons between WD and CCI at each time-
point were made using an unpaired i-test. Outliers in each group were determined using Dixon's test. Lesion
volume 2 weeks after WD trauma was compared with previously published values for 2-week posttraumatic
lesion volume after moderate CCI trauma (4.0 m/sec velocity, 2.5 mm depth) (Palmer et al., 1994) using an un-
paired Mest (the same CCI device and an identical severity of injury were used for this and the previous study).
A p value less than 0.05 was considered to be statistically significant.

RESULTS

All rats survived after trauma until the predetermined experimental timepoint was reached. Physiologic
variables, including arterial blood gas tensions, mean arterial blood pressure, rectal temperature, and hemat-
ocrit, were measured in all rats 15 min before trauma. No wifhin-group differences were found in any physio-
logic variable in the WD or CCI group (Table 1). A between-model comparison was significant only for a dif-
ference in Po2 (all WD = 170 ± 1.7, all CCI = 143 ± 2.9, p < 0.05). One rat in each of the 2 h and 24 h WD
groups had extreme values for MPO activity relative to the other values in their groups (0.3923 and 2.2501 U/g,
respectively). These values were determined to be statistical outliers and were not included in the data pre-
sented. Of note, these rats had large intraventricular and extraparenchymal hemorrhages, with upward hernia-
tion through the craniotomy on removal of the bone flap.
MPO activity as a function of time posttrauma is shown in Figure 1. MPO activity was below the detectable
limits of the assay in the control animals (-0.0071 ± 0.0041 and -0.0082 ± 0.0082 U/g, right and left hemi-
spheres, respectively) and in the nontraumatized hemisphere in all groups. MPO activity in the traumatized
hemisphere was increased at 24 and 48 h in both the WD (0.3152 ± 0.0472 and 0.3017 ± 0.0228 U/g, respec-
tively,/? < 0.05 vs controls) and CCI (0.1866 ± 0.0225 and 0.1937 ± 0.0772 U/g, respectively,/? < 0.05 vs con-
trols) models. At 24 h, MPO activity was 40.8% higher (p = 0.049), and at 48 h, it was 35.8% higher (p =
0.228) after WD than after CCI. MPO activity was not increased at 2 or 168 h in either model.
MPO activity at 24 h after CCI increased with the depth of injury (Fig. 2) (0.1866 ± 0.0225 vs 0.3011 ±
0.0141 U/g, 2.5 mm vs 3.0 mm depth, respectively, p < 0.05). With a 3.0 mm depth of injury after CCI, MPO
activity at 24 h posttrauma was similar to that observed at 24 h after WD (Figs. 1,2).
The sensitivity of this assay determined from the standard curve was 1.2 ± 0.3 x 10"4 U of MPO activity,
which represents the amount of MPO activity in approximately 2000 neutrophils. This corresponds to the
amount of MPO activity that would be seen in a 0.1 mL aliquot of supernatant from a typical 300 mg sample of
brain homogenate had there been 1.7 x 105 neutrophils/g brain tissue. In this study, we observed approxi-
mately 1-1.5 x 106 neutrophils/g brain tissue at 24 and 48 h posttrauma.

Table 1. Physiologic Data in Rats at 15 min Before Trauma

Group" Temp (°C) MAP HCT pH Pco2 Po2

WDb
2 h 37.2 +0.13e 94 ±4.3 42+1.7 7.44 + 0.02 37+1.2 168 + 4.4
24 h 37.0 ± 0.20 109 ± 8.3 41 ± 1.2 7.46 ± 0.00 37 ± 0.7 172 1 3.2
48 h 36.8 + 0.18 88 13.2 41+0.7 7.44 + 0.02 40+1.8 167 + 2.1
168 h 36.8 + 0.15 95 ± 2.9 42 + 0.8 7.44 10.02 40 11.5 175 13.3
All 36.9+0.09 9613.0 4110.5 7.44 10.01 39 10.7 17011.7*
CCI
2h 37.3 10.21 96 13.8 40 11.4 7.4110.01 4111.3 148 110.7
24 h 36.9 10.17 10114.7 4111.0 7.42 10.03 40 12.1 137 13.5
48 h 37.110.19 85 15.0 40 11.0 7.44 10.02 39 12.1 14113.1
168 h 37.0 10.13 95 16.1 43 11.4 7.43 10.02 40 10.8 144 12.6
All 37.0 1 0.09 94 1 2.7 41 1 0.6 7.42 1 0.00 40 1 0.8 143 1 2.9*

an = 4 per group.
bNo significant difference within WD or CCI groups by one-way ANOVA.
cMean 1 SEM.
*p < 0.05 between models by unpaired f-test.
 0.40

0.30
£
O)

0.20
È>
i-
o 0.10
<
o
D.
0.00

-0.10
CONTROL 2h 24h 48h 168h
Time Posttrauma
FIG. 1. Time course of neutrophil accumulation in brain (MPO activity in a 6-mm coronal section through the lesion) in
weight drop (hatched bars, n = 4/group), controlled cortical impact (solid bars, n = 4/group), and control rats (open bar, n =
4). MPO activity was defined as the degradation of 1 pmol of H202/min and was quantitated spectrophotometrically by
measuring the change in absorbance over 2 min at 460 nm. *p < 0.05 vs control by one-way ANOVA and Student-
Newman-Keuls test.

0.40

0.30 h
E

0.20
È>
O 0.10
<
O
a.
0.00

-0.10
CONTROL 2.5 3.0
Depth of Trauma (mm)
FIG. 2. Neutrophil accumulation (MPO activity) in brain at 24 h after moderate (4 m/sec velocity, 2.5 mm depth, n = 4),
and severe (4 m/sec velocity, 3.0 mm depth, n = 3) controlled cortical impact. MPO activity was defined as the degradation
of 1 pmol of H202/min and was quantitated spectrophotometrically by measuring the change in absorbance over 2 min at
460 nm. *p < 0.05 vs control (n = 4). #p < 0.05 3.0 mm depth vs 2.5 mm depth by one-way ANOVA and Student-Newman-
Keuls test.
 Lesion volume 2 weeks after WD trauma was 16.0 ± 2.7 mm3. Damaged regions of the brain typically in-
volved the parietal cortex, corpus callosum, hippocampus, and thalamus.

DISCUSSION

Neutrophil accumulation in brain, as measured by MPO activity, is increased at 24 and 48 h and resolves by
7 days in both the WD and CCI models. These results are consistent with those of Horner et al. (1992), who
demonstrated a similar time course in an analogous WD model (10 g weight dropped 10 cm) and confirmed
that the increase in MPO activity corresponded to the accumulation of neutrophils using immunohistochem-
istry. We have reported previously that MPO activity is a sensitive and specific method of quantitating neu-
trophil accumulation in brain after WD trauma by measuring MPO activity at 24 h postinjury in neutrophil-de-
pleted and nonneutropenic rats (Biagas et al., 1992b). Additional studies in our laboratory using labeled
neutrophils and erythrocytes showed that neutrophil accumulation in the brain between 0 and 2 h after trauma
was caused by hemorrhage associated with impact, whereas neutrophil accumulation between 4 and 8 h repre-
sented the onset of acute inflammation (Schoettle et al., 1990). Based on this previous work and the results of
the current study, the time course of neutrophil accumulation after brain trauma can be described as follows:
(1) immediately posttrauma (0-2 h), neutrophils are associated with hemorrhage, (2) between 4 and 8 h, accu-
mulation associated with inflammation begins, (3) at 24-48 h, accumulation is increased, and (4) by 7 days,
neutrophil accumulation resolves (Fig. 1).
A previous study from our laboratory (Palmer et al., 1994) reported a 2 week posttraumatic lesion volume of
13.2+ 1.7 mm3 in this same CCI model after moderate injury (4.0 m/sec velocity, 2.5 mm depth, 50 msec du-
ration of brain deformation). Compared with the present study, lesion volume in the CCI model was 17.5%
smaller than WD ( 16.0 ± 2.7 mm3). However, based on sample size and variability, this difference represented
only a trend and did not reach statistical significance. Peak MPO activity was 40.8% higher in the WD model
than in the CCI model. Thus, using a between-model comparison, more neutrophil accumulation (MPO activ-
ity) was also associated with a larger final lesion volume.
This study demonstrates the acute inflammatory response in two distinct models of cerebral trauma.
Although the dynamics of the models are different, lower velocity and longer duration of deformation in the
WD model vs higher velocity and shorter duration of deformation in the more contemporary CCI model, both
models as described produce a contusion (Schoettle et al., 1990; Palmer et al., 1993). Thus, it is not surprising
that the WD and CCI models produce a similar time course of neutrophil accumulation. Increasing the severity
of the CCI trauma by increasing the depth of penetration presumably leads to a larger contusion. Because MPO
activity increased with the depth of injury, this suggests that the amount of inflammation represented by neu-
trophil accumulation increases with the severity of trauma. This finding has been seen also in percussive spinal
cord injury (Xu et al., 1990). Additional studies are required to determine if models that primarily produce dif-
fuse injury without contusion, such as the midline fluid percussion model (Dixon et al., 1987) or the closed-
head weight drop model described by Montasser et al. (1994), will produce quantifiable posttraumatic neu-
trophil accumulation.
Neutrophils are important in nontraumatic models of brain injury. In models of cerebral ischemia, neu-
trophil accumulation has been demonstrated at 24 h by the MPO assay and histologie methods (Barone et al.,
1991), and recently, novel and selective antineutrophil therapies have been shown to have beneficial effects
(Clark et al., 1991 ; Chen et al., 1993). In rabbit meningitis models, selective inhibition of neutrophil adhesion
has been shown to reduce cerebrospinal fluid leukocytosis, blood-brain barrier damage, edema, and mortality
(Tuomanen et al., 1989; Granert et al., 1994).
The time course of neutrophil accumulation after trauma parallels important outcome variables of head in-
jury both in animal models and in humans, including alterations in cerebral blood flow, edema, and intracranial
hypertension (Obrist et al., 1984; Schoettle et al., 1990; Biagas et al., 1992a; Uhl et al., 1994). Specifically,
MPO activity and all of these cerebrovascular disturbances generally peak between 24 and 48 h posttrauma.
Although we have been unable to demonstrate an effect of neutrophil depletion on posttraumatic edema, we
have reported a reduction in posttraumatic hyperemia with vinblastine-induced neutropenia in the WD model
(Uhl et al., 1994). Accumulated neutrophils may play a similar role in the CCI model. The effects of novel and
selective antineutrophil therapies after trauma, such as those tested in nontraumatic models of cerebral injury
(Tuomanenetal., 1989; Clark et al., 1991; Chen et al., 1993;Granertetal., 1994), are currently under investi-
gation.
In conclusion, these data demonstrate the time course of neutrophil accumulation, as measured by MPO ac-
tivity, after WD and CCI trauma in rats. Specifically, ( 1 ) in both models, MPO activity after traumatic brain in-
jury is increased at 24 and 48 h and resolves by 168 h, (2) MPO activity in the CCI model increases with the de-
gree of trauma, and (3) posttraumatic neutrophil accumulation quantified by MPO activity is not a
phenomenon unique to the WD model.

ACKNOWLEDGMENTS

The authors would like to thank Dr. Joseph Carrillo for his critical review of this manuscript, Lisa Cohn for
her editorial assistance, and Dr. Heidi Horner (Athena Neurosciences) and Dr. Richard Stiller for their input re-
garding the myeloperoxidase assay. This work was supported in part by a seed grant from the University of
Pittsburgh Department of Anesthesiology and Critical Care Medicine. Dr. Kochanek is supported in part by an
Established Investigator Grant from the Society of Critical Care Medicine. Dr. Clark is supported in part by a
Fellowship Grant from the American Heart Association, Pennsylvania Affiliate.

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