JOURNAL OF NEUROTRAUMAVolume 19, Number 6, 2002© Mary Ann Liebert, Inc.

Effect of Posttraumatic Hyperglycemia on Contusion Volumeand Neutrophil Accumulation after Moderate Fluid-Percussion

Brain Injury in Rats

KOSAKU KINOSHITA,1,2 SUSAN KRAYDIEH,1,2 OFELIA ALONSO,1,2

NARIYUKI HAYASHI,3 and W. DALTON DIETRICH1,2

ABSTRACT

The purpose of this study was to evaluate the effects of posttraumatic hyperglycemia on contusionvolume and neutrophil accumulation following moderate traumatic brain injury (TBI) in rats. Aparasagittal fluid-percussion (F-P) brain injury (1.8–2.1 atm) was induced in male Sprague-Dawleyrats. Rats were then randomized into four trauma groups (n 5 7/group) by the timing of dextroseinjection (2.0 gm/kg/ip), which included (1) early (E) group: 5 min after TBI; (2) delayed (D) group:4 h after TBI; (3) 24-h group: 24 h after TBI; or (4) control (C) group: no dextrose injection. Asham operated control group also received dextrose to document physiological parameters (n 5 4).Rats were perfusion fixed 3 days following TBI, and the brains were processed for routine histopatho-logical and immunocytochemical analysis. Contusion areas and volumes, as well as the frequencyof myeloperoxidase immunoreactive polymorphonuclear leukocytes (PMNLs) were determined. Dex-trose injections significantly increased blood glucose levels (p , 0.005) in all treated groups. Al-though acute hyperglycemia following TBI did not significantly affect total contusion volume, con-tusion area was significantly elevated in the early treatment group. In addition, early posttraumatichyperglycemia enhanced neutrophil accumulation in the area of the cortical contusion (p , 0.005).In contrast, delayed induced hyperglycemia (i.e., 4 h, 24 h) did not significantly affect histopatho-logical outcome or neutrophil accumulation. Taken together, these findings indicate that acute butnot delayed hyperglycemia aggravates histopathological outcome and increased accumulation ofPMNLs. Posttraumatic hyperglycemia in the acute phase may worsen traumatic outcome by en-hancing secondary injury processes, including inflammation.

Key words: fluid-percussion; hyperglycemia; inflammation; neutrophil accumulation; secondary injury;traumatic brain injury

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1Departments of Neurological Surgery, University of Miami School of Medicine, Miami, Florida.2The Miami Project to Cure Paralysis, University of Miami School of Medicine, Miami, Florida.3Departments of Emergency and Critical Care Medicine, Nihon University School of Medicine, Tokyo, Japan.

INTRODUCTION

THE IMPORTANCE OF ISCHEMIC HYPERGLYCEMIA is wellestablished in the clinical and experimental settings

(Kent et al., 2001). In contrast, while clinical evidenceindicates that high blood glucose levels following TBIcan be associated with poor neurological outcome (Lamet al., 1991; Merguerian et al., 1981; Michaud et al., 1991;

Rovlias et al., 2000), the importance of blood glucose lev-els in the pathophysiology of neuronal damage after TBIhas not been clarified. Because hyperglycemia is commonlyobserved in patients following TBI (Young et al., 1989;Marmarou, 1992), the importance of blood glucose on trau-matic outcome after experimental TBI requires investiga-tion. In one study, pretrauma hyperglycemia was reportednot to affect intracellular pH or neurologic outcome aftermoderate F-P injury, an insult that does not result in en-ergy failure (Vink et al., 1997). However, following severecortical impact injury where primary ischemia occurs at thesite of impact, glucose administration increased contusionvolume (Cherian et al., 1998a).

Hyperglycemia has been reported to have deleteriouseffects on both global and focal cerebral ischemia (Gis-selsson et al., 1999; Siesjo et al., 1996). Pre- or postis-chemic hyperglycemia increases neuronal injury aftercardiac arrest (Myers and Yamaguchi, 1971), as well asfollowing transient periods of forebrain ischemia (Kalimoet al., 1981; Pulsinelli et al., 1982; Smith et al., 1988).In models of permanent focal ischemia, most studies havealso reported that hyperglycemia increases final damage,but published results are variable (de Courten Meyers etal., 1988; Ginsberg et al., 1987; Kraft et al., 1990; Ned-ergaard and Diemer, 1987; Zasslow et al., 1989). In mod-els of transient focal ischemia, both no effect (deCourtenMeyers et al., 1989, 1994) as well as increased tissuedamage have been reported (Nedergaard, 1987b; Robert-son et al., 1992). Variables, including glucose levels, in-sult duration, and degree of energy failure, may help ex-plain variable findings (Warner et al., 1995). Mechanismsunderlying aggravated damage are multifactorial (Siesjoet al., 1996). Increased formation of lactate and of H1

resulting in a decrease of intracellular or extracellular pHis an established consequence of ischemia (Myers, 1979;Nordstrom et al., 1976). Increased levels of lactate (Fol-bergrova et al., 1992; Harik and LaManna, 1988; Wag-ner et al., 1992) may also affect glial and capillary en-dothelial cells, leading to vascular perturbations (Kraiget al., 1986; Hurn et al., 1991; Plum, 1983; Siesjo et al.,1985). An effect on iron-mediated production of reactiveoxygen species has also been reported after hyper-glycemic ischemia (Hurn et al., 1991; Siesjo et al., 1985).

Previous studies have also demonstrated that hyper-glycemia causes a variety of pathological changes insmall vessels, arterioles, and peripheral nerves. Vascularendothelial cells are known to be an important target ofhyperglycemic damage (Nishikawa et al., 2000; Pieper etal., 1995; Kawai et al., 1997), but mechanisms underly-ing this damage to the cerebral microvasculature are notfully understood. Preischemic hyperglycemia has beenreported to aggravate blood–brain barrier (BBB) perme-ability following transient cerebral ischemia in rats

(Dietrich et al., 1993). Also, nuclear factor–kappaB (NF-kB), a critical cellular signaling molecule, has been im-plicated in the pathophysiology of hyperglycemia- or di-abetes-induced endothelial cell injury (Pieper andRiaz-ul-Haq, 1997).

Inflammatory processes are believed to contribute tothe pathogenesis of brain injury through secondary injurymechanisms (Kochanek et al., 1992; Clark et al., 1994;Feuerstein et al., 1994, 1997; Hartl et al., 1997; Jean etal., 1998; Grady et al., 1999; Chatzipanteli et al., 2000).The recruitment of polymorphonuclear leukocytes(PMNLs) into the injured brain can affect neuronal dam-age by various mechanisms, including the release of oxy-gen radical metabolites and hydrolytic enzymes, as wellas various cytokines (Tonnesen et al., 1988; Schoettle etal, 1990). Thus, previous studies concluded that neu-trophil accumulation in the early postinjured brain is as-sociated with increased neuronal damage and poor out-come (Clark et al., 1996; Hudome et al., 1997; Lin et al.,2000). In reference to hyperglycemia, Lin and colleagues(2000) have reported that preischemic hyperglycemialeads to enhanced PMNL accumulation following tran-sient global ischemia. Taken together, these studies indi-cate that postinjury hyperglycemia may adversely affecttraumatic outcome by enhancing PMNL accumulation.

The purpose of this study was to determine the effectof posttraumatic hyperglycemia in a reproducible modelof F-P brain injury. For these studies, we tested the ef-fects of induced hyperglycemia in both the acute andmore delayed postinjury periods. In addition to evaluat-ing contusion area and volume, the overall frequency ofPMNL accumulation was also quantitatively determinedby utilizing myeloperoxidase immunocytochemistry, asensitive marker of neutrophil accumulation (Chatzipan-teli et al., 2000).

MATERIALS AND METHODS

Animals

The protocol for these animal studies was approved bythe University of Miami Animal Care and Use Commit-tees. The histopathological studies were performed on 32male Sprague-Dawley rats weighing 260–335 g obtainedfrom Charles River Breeders. All animals were kept at aconstant temperature (23–25°C) in an air-conditionedroom for at least 7 days before the study and exposed toa 12-h light-dark cycle. Rats were allowed free access towater, but food was withheld overnight before surgery.

Traumatic Brain Injury

The basic surgical preparation for the F-P brain injurywas performed according to methods previously de-

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scribed (Dietrich et al., 1994). On the following day, aF-P device was used to produce experimental TBI (Dixonet al., 1987; McIntosh et al., 1996). The rats were ini-tially anesthetized with 3.0% halothane in a gas mixtureof 70% nitrous oxide and 30% oxygen. Femoral arteryand vein were cannulated with PE-50 tubing. Rats werethen immobilized with pancuronium bromide (0.5 mg/kg,iv). An endotracheal tube was inserted orally, and ratswere mechanically ventilated on a mixture of 70% ni-trous oxide, 0.5–1.5% halothane with a balance of oxy-gen. Rats were then placed in a stereotaxic frame, and a4.8-mm craniotomy was made overlying the right pari-etal cortex (3.8 mm posterior to bregma and 2.5 mm lat-eral to the midline; Zilles, 1985). A plastic injury tubewas then placed over the exposed dura, was bound byadhesive as well as dental acrylic and hardened, and thescalp was closed. Next, the F-P injury device was usedthrough the injury tube to create an experimental TBI.Prior to undergoing TBI, the rats were monitored withrespect to blood gases, mean arterial blood pressure,hematocrit, and blood glucose level. As an indicator ofbrain temperature, a thermometer (30-gauge; OmegaNeedle Probe, Stamford, CT) was inserted in the left tem-poralis muscle. Rectal and brain temperatures were main-tained at normothermic levels (37°C) before TBI.

Hyperglycemia

The animals were randomly assigned to receive an in-traperitoneal injection of 2.0 g/kg (0.5 g/mL) dextrose withminor modifications of previous publication (Dietrich etal., 1993). This method of increasing blood glucose levelsdoes not significantly alter serum osmolality. Sham-oper-ated animals (n 5 4) were fasted overnight and receivedan intraperitoneal injection of 2.0 g/kg dextrose for all sur-gical procedures except actual F-P injury, in order to in-vestigate temporal profile of physiological parameters af-ter injection of dextrose. Temporal muscle temperature(TMT), mean arterial blood pressure (MAP), arterial pH,PaCO2, PaO2, hematocrit, and blood glucose were mea-sured prior to the injection of dextrose, as well as 15 and30 min, and 1, 2, 3, and 4 h after injection of dextrose.

This study included four trauma groups (n 5 7 pergroup): (1) early dextrose injection group (E group), inwhich the animals received dextrose at 5 min after TBI;(2) delayed dextrose injection group (D group), where an-imals received dextrose 4 h after TBI; (3) 24 h after TBIgroup (24 h group), where animals received dextrose 24 hafter TBI; (4) control group (C group), where rats under-went all surgical procedures including F-P injury but re-ceived a comparable volume of saline. Thirty minutes af-ter TBI, all animals were returned to their cages to recover.

TMT, MAP, arterial pH, PaCO2, PaO2, hematocrit, and

blood glucose were measured at the period of the prein-jection of dextrose, and 15 min or 4 h after the injectionof dextrose. Femoral artery catheters were left in place toobtain blood samples. At 4 h after TBI, animals were re-anesthetized with halothene and a balance of oxygen witha mask for the purpose of taking blood samples. Food wasprovided to all animals at 4 h after TBI, except for the Dgroup. The animals of D group received intraperitoneal in-jections of dextrose 4 h after TBI. At 8 h after TBI, theanimals were reanesthetized and blood samples taken. Allanimals of D group were given food and water ad libitum4 h after injection of dextrose (8 h after TBI). The animalsin the 24-h group were given an intraperitoneal injectionof dextrose under mask anesthesia 24 h after TBI.

Histopathological Studies

At 3 days after TBI, the animals were deeply reanes-thetized and were perfusion-fixed transcardially with coldphysiologic saline for 2 min and then FAM (a mixture of40% formaldehyde, glacial acetic acide, andmethanol; 1:1:8by volume) for 20 min (25°C at a pressure of 100–120 mmHg). Brains were extracted and stored in chilled fixativeovernight. The next day, brains with the dura attached wereembedded in paraffin and10-mm coronal sections obtainedat various levels (1.8, 2.3, 3.3, 3.8, 4.8, 5.8, 6.8, and 7.3mm posterior to bregma; Zilles, 1985) were used to obtaincontusion area measurements (Dietrich et al., 1994). Hema-toxylin and eosin–stained sections were prepared for mea-surement of contusion area and volume (n 5 7 per eachgroup). The cortical contusion boundaries were well de-marcated, and represented an area of shearing damage be-tween the cortex and external capsule and consisted of py-knotic neurons, reactive astrocytes, and abnormal whitematter tracts. The tissue also appeared hemorrhagic in someareas, and the extracelluar space was edematous relative toregions outside of the contused area.

To determine the areas and volumes of tissue contu-sion, lesions were traced at a power of 1003 by using acamera lucida microscope attachment. The contused ar-eas were calculated by retracing these drawings onto adigitizing tablet that was interfaced with a computer. Thecontusion volume was computed using numerical inte-gration of sequential areas.

Selective sections were also prepared for neutrophilcounts using myeloperoxidase (MPO) immunocyto-chemistry (n 5 7/group). Brain coronal sections were re-hydrated and placed in 6% H2O2 to block endogenousperoxidase activity. The tissue was rinsed and steamedwith a solution of citrate buffer for 20 min and rinsedwith 0.05 mol/L phosphate-buffered saline. Nonspecificactivity was blocked with normal horse serum. Subse-quently, brain sections were incubated at room tempera-

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ture for 1 h in a 1:15,000 dilution of rabbit antihumanMPO (DAKO Corp., Carpenteria, CA). To test for non-specific staining, negative controls were conducted wherethe primary antibody was omitted during tissue process-ing. Further rinsing was done with phosphate-bufferedsaline, the secondary antibody was applied, and the slideswere processed using the LSBA staining kit (DAKOCorp.) per the manufacturer’s instructions. MPO stainingwas visualized with peroxidase, and diaminobenzidinewas used as a chromogen to yield a brown reaction prod-uct. Slices were washed in 0.5% Triton X-100, followedby 1% cupric sulfate to further intensify staining. Neu-trophils were counted manually by an investigatorblinded to the experimental groups using microscopicevaluation (2503) of randomly selected fields (0.16mm2). These fields included the cortical lesion from brainsections at selective brain levels, including 1.8, 2.3, 3.3,

3.8, 4.8, 5.8, 6.8, and 7.3 mm posterior to bregma. Neu-trophil counts (0.16 mm2) at each brain section, as wellas mean numbers of neutrophils in all sections for the ex-perimental groups, were then compared.

Statistical Analysis

Data were expressed as mean values 6 SEM. Analy-sis of variance (ANOVA) for repeated measures was usedto compare physiologic variables measured over time.Between group comparisons of physiological data weremade using ANOVA one-way analysis of variance. Fornonparametric data, Kruskal-Wallis one-way ANOVA onranks was used. Post hoc comparisons were made usingthe Student-Newman-Keuls test for parametric data orDunn s method for nonparametric data. Differences wereconsidered significant if p value was ,0.05.

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FIG. 1. Blood glucose levels after intraperitoneal injection of dextrose in sham-operated animals. Increased levels of blood glucosewere observed within 15 min after injection of dextrose, as compared with preinjection levels. ***p , 0.001 at 15 min, 30 min, 1 h,and 2 h; **p , 0.005 at 3 h; *p , 0.05 at 4 h (one-way ANOVA repeated measured followed by Students-Newmann-Keuls test).

TABLE 1. BASELINE PHYSIOLOGICAL VARIABLES BEFORE TBI

C group E group D group 24-h group

TMC (°C) 36.7 6 0.07 36.8 6 0.1 36.6 6 0.1 36.9 6 0.1PCO2 (mm Hg) 38.0 6 0.5 38.3 6 0.7 38.0 6 0.6 38.0 6 0.8PO2 (mm Hg) 139.0 6 8.4 122.9 6 4.2 124.3 6 9.2 140.3 6 11.7pH (unit) 7.454 6 0.013 7.453 6 0.01 7.466 6 0.008 7.444 6 0.007MAP (mm Hg) 115.1 6 2.8 112.0 6 2.8 121.3 6 2.9 119.3 6 2.3Ht (%) 47.4 6 1.4 48.6 6 0.5 46.1 6 1.2 46.3 6 0.8Blood glucose (mg/dL) 129.7 6 4.7 130.0 6 6.9 141.9 6 7.6 144.1 6 8.6

Values are mean 6 SEM. There was no significant difference between groups. TMT, temporalis muscle temperature, MABP,mean arterial blood pressure; Ht, hematocrit.

RESULTS

Physiological Parameters

Baseline data. Physiological parameters were obtainedfor each group at 15 min before, and 15 min or 4 h afterTBI and the injection of dextrose. The baseline physio-logical variables are presented (Table 1). No significant

difference was observed between groups in TMT, PaCO2,PaO2, arterial pH, MAP, hematocrit, or glucose.

Dextrose injections. Figure 1 indicates the changes inblood glucose levels after the intraperitoneal injection ofdextrose in sham-operated animals. There was a signifi-cantly increase in blood glucose from 15 min to the end

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FIG. 2. Physiological parameters after dextrose injection. There were significant differences in PaCO2 (A) at 15 min and MAP(D) at 4 h between the four treatments groups. Hematocrit level (E) significantly differed between preinjection and 15 min and4 h (**p , 0.005, *p , 0.05 following one-way ANOVA post hoc Student-Newman-Keuls).

of the observational period (4 h after dextrose injection).Blood glucose levels peaked at 15 min (499 6 20.0mg/dL) after injection of dextrose and remained elevatedafter 1 h (305.0 6 19.8 mg/dL), and then gradually re-turned to baseline. However, blood glucose remained sig-nificantly elevated (p , 0.05) at 4 h after injection(192.8 6 9.4 mg/dL) as compared to baseline.

Figure 2 indicates the physiological parameters 15 minand 4 h after injection of dextrose in each experimentalgroup. There were significant changes in PaCO2 at 15min (Fig. 2A; p , 0.05, D group vs. sham, and C, and Egroup, 24-h group vs. sham.), and MAP at 4 h (Fig. 2D;p , 0.05, sham vs. D group) after dextrose injection (one-way ANOVA post hoc Student-Newman-Keuls). Hema-tocrit levels significantly differed (Fig. 2E; p , 0.005) atpreinjection, 15 min (24-h group vs. all other groups),and 4 h after injection of dextrose (24-h group vs. allother groups, D group vs. sham and E group).

Figure 3 indicates the blood glucose level after injec-tion of dextrose at various time periods following TBI.Although no significant changes in glucose levels wereobserved at baseline (Table 1), glucose levels were sig-nificantly increased at the preinjection period in the 24-h group (p , 0.001). There were significant increases inblood glucose level at 15 min and 4 h after injection ofdextrose in C group as compared to all other groups (p ,

0.001). The F-P-injury had no effect on blood glucoseconcentration, and the blood glucose levels remainedclose to fasting baseline values at 15 min, 4 h after TBI(Table 1 and Fig. 3, C group).

Histopathological outcome. In the early (E) 5-mintreatment group, the mean contusion area was signifi-cantly (p , 0.05) increased at bregma level 4.8 as com-pared with other TBI treatments groups (one-wayANOVA using Student-Newman-Keuls post hoc test).However, no significant changes were observed betweenthe four treatment groups at other bregma levels (Fig. 4).

The mean contusion volume in the C group was 1.45 6

0.19 mm3 compared with 1.59 6 0.19 mm3 in the Egroup, 1.19 6 0.26 mm3 in the D group, and 1.18 6 0.23mm3 in the 24-h group. Analysis of the contusion vol-ume data demonstrated no significant difference betweengroups following one-way ANOVA.

Figure 5 represents the number of MPO-positive neu-trophils obtained from cell counting in the injured corti-cal sections. The number of neutrophils/0.16 mm2 wassignificantly increased in injured cortex in only the Egroup at the brain level of 4.8 mm posterior to bregmaas compared to other treatments groups (p , 0.05; one-way ANOVA using Student-Newman-Keuls post hoctest). There were no significant changes in neutrophil

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FIG. 3. Changes in blood glucose levels after dextrose injection at various time points following TBI. Blood glucose level be-fore injection of dextrose (***p , 0.001, 24-h group vs. all other groups) significantly elevated in the 24-h group. There was asignificant increase in blood glucose 15 min and 4 h after injection of dextrose in C-group as compare to all other groups (***p ,

0.001 following one-way ANOVA post hoc Student-Newman-Keuls).

counts at 5.8 (p 5 0.055) or 6.8 mm (p 5 0.052) poste-rior to bregma as compared to C group. The mean num-ber of neutrophils/0.16 mm2 at all sections (Fig. 6) wasalso significantly increased in injured cortex in the Egroup, when compared with other treatment groups (p ,

0.05; one-way ANOVA using Student-Newman-Keulspost hoc test).

DISCUSSION

These data demonstrate that acute hyperglycemia af-ter moderate F-P injury leads to enhanced neutrophil ac-cumulation in the cortical contusion area. This neutrophilaccumulation was associated with increased contusionarea. In contrast, hyperglycemia initiated at a late phasefollowing TBI had no effect on neutrophil accumulationor histopathological outcome. These results are of par-ticular interest, because neutrophils may contribute tosecondary injury after ischemia/reperfusion by causingmicrovascular occlusion (del Zoppo et al., 1991) and re-leasing oxygen radicals, cytolytic proteases, and proin-flammatory cytokines (Feuerstein et al., 1994; Hartl etal., 1996), which, in addition to direct neuronal damage,may injure the endothelium.

Previous TBI studies have reported variable findingswith pre- and post-hyperglycemia, depending on the in-

jury model tested. Thus, while no adverse effect on out-come was reported after moderate F-P brain injury (Vinket al., 1997), glucose infusion significantly increased con-tusion volume and neuron density in CA1 and CA3 re-gions of the hippocampus following mild cortical impactinjury (CCI) complicated by a secondary ischemic insult(Cherian et al., 1998b). In another study by this group,pre- but not posttraumatic hyperglycemia increased con-tusion volume after severe CCI injury (Cherian et al.,1998a). The present findings indicate that acute post-traumatic hyperglycemia also worsens histopathologicaloutcome after F-P brain injury. In the clinical setting, arapid and large increase in blood glucose concentrationis a frequent event in patients with severe brain injury(Michaud et al., 1991; Lam et al., 1991). Thus, there re-main some questions regarding whether patients with se-vere TBI should receive glucose-containing IV fluids asmaintenance fluids.

The present experiment focused on histopathologicaland inflammatory changes in response to induced acutehyperglycemia at various time points following moder-ate TBI. Previous studies indicate that a chronic diabeticstate induces an exaggerated inflammatory response toischemia-reperfusion manifested as a greater accumula-tion of leukocytes and increased albumin extravasationin peripheral vessels (Panes et al., 1996). However, thereis less information regarding whether acute hyper-

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FIG. 4. Mean contusion areas (mm2) following TBI. Significantly (*p , 0.05) increased mean contusion area at 4.8 mm pos-terior to bregma in E-group as compared with the other treatment groups.

glycemia alone affects inflammation following TBI. Ad-ditionally, it has been suggested that acute hyperglycemiaincreases the production of reactive oxygen species (Gi-ardino et al., 1996; Ceriello, 1997), and previous studieshave demonstrated that high glucose and free radicals in-duce the cellular expression of the endothelial expression

molecule (ICAM-1; Ceriello et al., 1996, 1998; Marfellaet al., 2000). In addition, it is well known that diabetes(Richardson et al., 1994; McLeod et al., 1995) and hy-perglycemia (Baumgartner-Parzer et al., 1995) alonecause increased expression of various adhesive proteins.One goal of this experiment was, therefore, to determine

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FIG. 5. Number of neutrophils/0.16 mm2 in the injured cortex at each bregma level 3 days after TBI. MPO-immunoreactiveneutrophils were counted manually with the aid of a microscope (2503). Numbers of neutrophils were significantly increased(**p , 0.005 at 4.8 mm posterior to bregma in E group, while there was no significant difference at other brain levels.

FIG. 6. The mean number of neutrophils/0.16 mm2 in each group. A significant (*p , 0.05) increase in the mean number ofneutrophils was observed in the E group compared with the other treatments groups following TBI (one-way ANOVA using Stu-dent-Newman-Keuls post hoc test).

whether posttraumatic hyperglycemia induced at varioustime points has an adverse effect on neutrophil accumu-lation following TBI.

Brain inflammation has been implicated in the patho-genesis of secondary brain damage (Kim, 1996; Feuer-stein, et al., 1997). Recently, several authors have re-ported that F-P brain injury leads to a pronouncedaccumulation of PMNLs. Neutrophil accumulation re-mains increased up to 24–48 h after trauma (Carlos et al.,1997; Royo et al. 1999; Whalen et al., 1999; Keeling etal., 2000; Chatzipanteli et al., 2000) and tends to declineat 7 days. Methods of limiting neutrophil infiltration, in-clude the reduction of circulating neutrophils (Matsuo etal., 1994), administration of antibodies against endothe-lial or leukocyte-expressed adhesion molecules (Choppet al., 1994), and use of ICAM-1 (Soriano et al., 1996),reduce infarct size and brain swelling. Our experimentsdemonstrate enhanced neutrophil accumulation withacute hyperglycemia in a moderate TBI model uncom-plicated by a secondary ischemic insult.

It has been proposed that hyperglycemia may con-tribute to endothelial cell damage in brain ischemia mod-els (Paljarvi et al., 1983; Dietrich et al., 1993; Siesjo etal., 1996; Li et al., 1997; Lin et al., 1998, 2000). Althoughthe exact mechanisms by which hyperglycemia mayworsen outcome are poorly understood, acute hyper-glycemia after TBI may facilitate endothelial cell dam-age, leading to the increased expression of endothelialadhesion molecules and the enhancement of neutrophilinfiltration into the injured brain. Further studies are re-quired to fully explore the complex roles of neutrophilaccumulation in the setting of acute hyperglycemia fol-lowing TBI.

CONCLUSION

Our experiment demonstrates that (1) TBI induces sig-nificant neutrophil accumulation within injured brainwith acute hyperglycemia and (2) acute, but not delayedhyperglycemia, influenced contusion area following TBI.These findings indicate that posttraumatic acute hyper-glycemia may create a condition whereby inflammatoryprocesses are magnified, rendering the severe head in-jured patient at high risk for secondary brain damage.

ACKNOWLEDGMENTS

This study was supported by National Institutes ofHealth grant NS30291. We thank Min Chen and XiaoShang for excellent technical assistance, and Charlaine

Rowlette for excellent editorial assistance and manuscriptpreparation.

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