Journal of Cerebral Blood Flow and Metabolism 19:11l8-1125 © 1999 The International Society for Cerebral Blood Flow and Metabolism Published by Lippincott Williams & Wilkins, Inc" Philadelphia

Interleukin-l Receptor Antagonist Attenuates Regional Neuronal

Cell Death and Cognitive Dysfunction After Experimental

Brain Injury

Kristin L. Sanderson, Ramesh Raghupathi, Kathryn E. Saatman, *David Martin, *Gerald Miller, and Tracy K. McIntosh

Department of Neurosurgery, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania; and *Department of

Pharmacology, Amgen, Inc., Thousand Oaks, California, U.S.A.

Summary: The effect of systemic administration of human recombinant interleukin-I receptor antagonist (rhIL-Ira) on be­havioral outcome and histopathologic damage after lateral fluid-percussion brain injury of moderate severity was evalu­ated. In study I, brain-injured Sprague Dawley rats received timed subcutaneous injections beginning IS minutes after in­jury of either 100 mg/kg rhIL-lra (high dose, total dose =

1900 mg/kg), 10 mg/kg rhIL-1 ra (low dose, total dose = 190 mg/kg), 6r vehicle over 7 days. No effect of low-dose rhTL-lra was observed in study 1. High-dose rhIL-I ra significantly at­tenuated posttraumatic neuronal loss in the injured hippocam­pal CA3 region (P < 0.05), dentate hilus (P < 0.05), and cortex (P < 0.05) but impaired recovery of motor function at 7 days after trauma (P < 0.05). In study 2, rats were pretrained to learn a visuospatial task in a Morris water maze, subjected to fluid-

Traumatic brain injury (TBI) is known to cause severe neurologic dysfunction in humans and animals. Posttrau­matic functional deficits have been associated with sec­ondary or delayed damage, which develops hours to days after the initial trauma. Several studies suggest that this secondary injury may be mediated, in part, by the ex­pression and release of specific cytokines, including the

Received October 8, 1998; final revision received February 25, 1999; accepted March I, 1999.

Supported in part by grants from the National Institutes of Health P50-NS08803 and ROl-NS26818 from NlNDS, ROI-GM34690 from NIGMS, a Veterans Administration Merit Review Grant, and a grant from Amgen Inc., Thousand Oaks, CA, U.S.A ..

Address correspondence and reprint requests to Dr. Tracy K. McIntosh, Department of Neurosurgery, University of Pennsylvania, School of Medicine, 105 Hayden Hall, 3320 Smith Walk, Philadelphia, PA 19104-4283, U.S.A.

Abbreviations used: ANOV A, analysis of variance; APP, amyloid precursor protein; FP, fluid-percussion; IL- I , interleukin-I; IL- I[3, in­terleukin-1[3; mRNA, messenger ribonucleic acid; NGF, nerve growth factor; rhIL-Ira, recombinant human interleukin- I receptor antagonist; TBI, traumatic brain injury.

1118

percussion brain injury or' sham treatment, and randomly as­

signed to receive multiple subcutaneous injections at timed intervals of 100 mg/kg rhIL-lra (total dose = 900 mg/kg) or

vehicle over 42 hours, followed by continuous infusion of a

lower concentration of rhIL-lra (20 mg/kg/day, total dose =

100 mg/kg) , or vehicle for 5 days using subcutaneously im­planted osmotic minipumps. Postinjury administration of rhIL­Ira significantly attenuated cognitive deficits compared with vehicle-treated animals at 42 hours (P < 0.05) but did not affect motor function at 48 hours, 1 week, and 2 weeks. These results suggest that inhibitors of cytokine pathways may be therapeu­tically useful for the treatment of brain trauma. Key Words: Traumatic brain injury-Cytokines-Interleukins­Histopathology-Behavior-Fluid-percussion.

interleukins such as interleukin-l � (IL-l �), interferons, and tumor necrosis factor-a (Rothwell et aI., 1993).

Although specific cellular sources of IL-J � in the CNS are unknown, activated microglia, neurons, and as­trocytes have been implicated. In addition, systemic IL-1� is transported across the blood-brain barrier by a saturable transport system (Gutierrez et aI., 1994). Inter­leukins such as IL-l � have been reported to play a role in neurodegeneration after TEl or ischemia (Rothwell, 1998). An exacerbation of ischemic brain damage has been observed after localized striatal injections of IL-l � in the rat (Stroemer and Rothwell, 1998). Patients with severe head injury have been shown to have elevated CSF levels of IL- I � (Young et aI., 1988), and experi­mental models of brain injury also are associated with increased levels of IL-l � throughout the injured cortex in the acute posttraumatic period (hours to 2 days) (Taupin et aI., 1993). Moreover, Northern blot analyses have revealed significantly increased gene expression (messenger ribonucleic acid [mRNA]) for IL-l� in in­jured hippocampus and cortex as early as 1 hour after

rhlL-1ra TREATMENT IN TBI 1119

fluid-percussion (FP) brain injury in the rat, which re­turned to undetectable levels by 24 hours (Fan et aI., 1995).

Interleukin- l [3 has been linked to the development of neuritic [3-amyloid plaques in Alzheimer's disease (Sheng et a!., 1996), and increased expression of IL- l has been correlated with elevated levels of [3-amyloid pre­cursor protein ([3APP) after human head injury, suggest­ing a possible role for interleukins in Alzheimer's disea­se-like neurodegenerative changes associated with brain trauma (Griffin et aI., 1994). Interleukin- I [3 has been reported to stimulate astroglial proliferation and gliosis in the injured brain (Scripter et aI., 1997) and has been implicated in the activation of inducible nitric oxide syn­thase (Lee et a!., 1995; Rothwell and Relton, 1993), lead­ing to accumulation of potentially pathologic levels of nitric oxide.

An endogenously occurring antagonist of interleu­kin-l (IL- l) receptors has been isolated from the condi­tioned medium of cultured human monocytes (Hannum et aI., 1990). Recombinant human lL- l receptor antago­nist (rhIL- I raj is a 17 -kd polypeptide, which is highly homologous to IL- la and IL-l[3 and competitively binds to IL- l type-I receptors (Hannum et a!., 1990). Intrace­rebroventricular administration of rhIL-l ra has been re­ported to reduce infarct volume in a variety of models of cerebral ischeinia (Garcia et aI., 1995; Relton et a!., 1996). Recently, the cortical lesion induced by FP brain injury has been shown to be reduced by repetitive postin­jury intracerebroventricular infusions of rhlL- lra (Toul­mond and Rothwell, 1995), whereas DeKosky and col­leagues report that cells transfected to express rhIL- lra, when transplanted into the brain, can attenuate trauma­induced increases in nerve growth factor (NGF) levels (DeKoskly et a!., 1996). Like IL-J [3, rhIL- lra crosses the blood-brain barrier through a transport system, although only 0.3% to 0.6% of a systemically administered dose per gram of brain tissue enters into the eNS (Gutierrez et aI., 1994). Pretreatment or posttreatment with zinc pro­toporhyrin, an IL- l receptor antagonist and hemeoxygen­ase inhibitor, also was reported to reduce total size and area of infarct after middle cerebral artery occlusion (Kadoya et a!., 1995; Yamasaki et a!., 1995), whereas infarct volume and striatal neuronal loss after experimen­tal hypoxia-ischemia have been shown to be reduced by subcutaneous injection of rhIL-lra in the neonatal rat (Martin et a!., 1994). The current study is the first ex­amination of the effects of systemically administered rhIL-lra on neurobehavioral and histologic outcome af­ter experimental TBI in the rat.

METHODS

Fluid-percussion injury A two-part study was conducted using adult male Sprague­

Dawley rats (n = 102, 348 to 422 g). Rats were anesthetized

with sodium pentobarbital (60 mg/kg) and mounted in a ste­reotaxic frame. A midline incision was made in the scalp, fol­lowed by a craniotomy (5-mm diameter) centered between lambda and bregma. A modified Luer-Lok cap was secured with dental cement into the craniotomy, over the intact dura. Lateral FP injury of the left hemisphere was induced, as ini­tially described (McIntosh et aI., 1989). Briefly, a calibrated saline-filled device was attached to the rat by the Luer-Lok cap, and a 25-millisecond pulse of saline was delivered under pres­sure to the intact dura to create a brain injury of moderate severity (study 1: 2.5 ± 0.1 atm, n = 45; study 2: 2.5 ± 0.1 atm, n = 27). Animals were monitored closely for respiratory dis­tress during the first 15 minutes after injury and allowed to recover on a heating pad. Sham (uninjured) rats were subjected to anesthesia and surgery without brain injury (study 1, n = 16; study 2, n = 14). Control and experimental groups were be­haviorally tested, injected, and killed at equivalent times.

Drug administration Study 1. Both rhIL-lra (Amgen, Inc., Thousand Oaks, CA,

U.S.A.) and vehicle (lO mmollL citrate, pH 6.5, 140 mmollL NaC, 0.05 mollL ethylenediamine tetraacetic acid) were stored at -20°C, then warmed to room· temperature at the time of injection. In study I, brain-injured animals were randomly se­lected to simultaneously receive an intravenous bolus and sub­cutaneous injection of vehicle, low-dose rhIL-l ra (10 mg/kg), or high-dose rhIL-l ra (100 mg/kg) at 15 minutes after injury. Doses were based on previously published studies (Martin et aI., J 994; Relton et aI., 1996). This was followed by repeated subcutaneous injections at 4,8, 12,20,24,32, and 44 hours and every 12 hours until day 7 after injury. Sham animals received either vehicle or high-dose rhIL-lra using an identical admin­istration paradigm.

Study 2. Brain-injured and sham animals received a similar simultaneous intravenous and subcutaneous bolus of either ve­hicle or high-dose rhlL-lra at 15 minutes after injury, followed by subcutaneous injections at 4, 8, 16, 20, 24, 28, and 40 hours. Although the exact timing of postinjury drug administration differed slightly between studies 1 and 2, the total dose of rhIL-lra (900 mg/kg) and volume of vehicle (9 mL/kg) up to 48 hours after injury was identical in the two studies. Since re­peated administration of high-dose rhIL-l ra between days 2 and 7 after injury in study 1 appeared to slightly retard the recovery of motor function, we decided to use the low dose (10 mg/kglinjection) between days 2 and 7 in study 2. However, in study 2, we also avoided the potential stress of the twice-daily injection schedule of study I by administering the low-dose rhIL-I ra and vehicle through a continuous infusion minios­motic pump (Alza, Inc., Wilmington, DE, U.S.A.). To implant these pumps, animals were reanesthesized (sodium pentobar­bital, 60 mg/kg intraperitoneally) at 48 hours after injury, and two Alzet pumps (lOOID) were implanted subcutaneously above the haunches of the rat. On day 7 after injury, animals were reanesthetized (sodium pentobarbital, 60 mg/kg intraper­itoneally), the pumps removed, and the contents analyzed to determine the amount of drug each animal received. The total dose of rhIL-lra (100 mg/kg) and the total volume of vehicle (10 mLlkg) received by brain-injured animals between days 2 and 7 after injury in study 2 were identical to those received by their study 1 counterparts in the same postinjury period.

Physiologic monitoring In a separate group of rats maintained under sodium pento­

barbital anesthesia (n = 8 brain-injured, n = 3 shams), tem­poral muscle temperature, core body temperature, mean arterial

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1120 K. L. SANDERSON ET AL.

blood pressure, and heart rate were monitored after brain in­jury/sham surgery and intravenous treatment with rhIL-l ra (100 mg/kg, n = 4) or vehicle (n = 4). Parameters were measured for 15 minutes before FP brain injury for baseline measurements, then every 15 minutes for 2 hours after injury and drug/vehicle administration. Core body temperature was recorded by insertion of a plastic coated thermocouple into the rectum, whereas temporal muscle temperature was monitored through placement of a temperature probe between the skull and the temporal muscle. Mean arterial blood pressure and heart rate were recorded with a Cardiomax II (Columbus In­struments, Columbus, OH, U.S.A.) through a catheter placed in the femoral artery.

Behavioral testing: motor function Animals in study I were evaluated for neurologic motor

function at 2 and 7 days after injury, whereas animals in study 2 were tested for motor function using identical tests at 2, 7, and 14 days after brain injury. Motor function tests were con­ducted by a trained investigator who was blinded to the treat­ment regimen and injury status of the animals and consisted of forelimb flexion, hindlimb flexion, lateral pulsion, and angle board tests. A score from 0 to 4 was given on the performance for each task (where 0 = afunctional and 4 = normal, unin­jured performance). The forelimb flexion test consisted of rapid introduction of a flat surface to the rat when suspended by the tail where the position of each of the forelimbs is recorded. The hindlimb flexion test examined the backward extension of each of the hindlimbs when the animal's tail was lifted and the forelimbs remained on a hard surface. Lateral pulsion evaluated the animal's ability to resist hand pressure when moved in either dir�ction. The angle board test evaluated the rat's ability to stand on an inclined plane in a right, left, and vertical posi­tion compared with a baseline performance (4 = above or equal to baseline; 3 = 2.5" below baseline; 2 = 5" below baseline; 1 = 7.5° below baseline; 0 = 10° or more below baseline). The angle board scores were averaged across the three positions and added to those from other tests to achieve a maximum composite score for motor function of 28 points.

Behavioral testing: cognitive function Performance of cognitive function was evaluated in study 2

using a visuospatial memory Morris water maze paradigm de­scribed previously (Smith et a!., 1991). One day before and on the day of injury, all animals were pretrained in the Morris water maze, a I-m diameter tank filled with water at 18°C. Animals were trained over a series of 20 trials (10 trials/day) to find a submerged platform after being placed at several differ­ent entry points in the Morris water maze, using visual cues located outside the maze. Latency from the starting point to the hidden platform was recorded for each trial to determine the learning ability of each rat. At 42 hours after sham treatment (n

= 14) or FP injury and treatment with either rhIL-lra (n = 13) or vehicle (n = 14), the platform was removed from the maze, and animals were evaluated for their ability to recall the plat­form location. The swim patterns were video recorded, and a computer-generated numerical score was calculated where more weight was assigned for time spent in regions closest to the former platform location (Smith et aI., 1991). The swim distance and average swim speeds also were calculated for each animal.

Histologic evaluation Animals were overanesthetized with sodium pentobarbital

(200 mg/kg) and intracardially perfused with a solution of 100

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mL of heparin/saline followed by approximately 300 mL of 10% buffered formalin (Sigma, St. Louis, MO, U.S.A.). Ani­mals from study 1 (n = 15, vehicle treated; n = 16, 10 mg/kg rhIL-I ra treated; n = 14, 100 mg/kg rhIL-lra treated; n = 16, sham) were perfused at 8 days after injury. Brains were re­moved immediately and postfixed in formalin for 24 hours.

After fixation, brains were dehydrated, embedded in paraf­fin, cut coronally in 5-J.Lm-thick sections, and stained with cre­syl violet. Using a Leitz microscope, viable neuronal counts were performed by a trained observer blinded to the treatment status of the animals. Regional areas were measured using a

100- mm2 eyepiece corresponding to 0.25 mm on a linear cali­brated scale using a 20x objective. Cell counts were expressed as the number of cells per given area. Brain regions were cho­sen in accordance with previous data indicating specific areas of cell loss in the traumatically injured brain (Hicks et aI., 1996). Neurons were defined based on the presence of an in­tensely stained nucleolus, unstained nucleus, and the presence of a cell body. Neuronal counts in the following 10 brain re­gions were obtained: two areas of the parietal cortex (areas 1 and 2, corresponding to the site of maximal injury and the periinjury "penumbra" region, respectively, at -3.3 mm bregma), temporal cortex, retrosplenial agranular cortex, CA3 region of the hippocampus, hilus of the hippocampal dentate gyrus and medial geniculate nucleus (all at -4.8 mm bregma), lateral septal nucleus (at 0.2 mm anterior to bregma), ventral posterolateral thalamic nucleus (at -3.3 mm bregma), and sub­stantia nigra (at -6.04 mm bregma).

Statistical analysis All neuroscore data were statistically examined using

Kruskal-Wallis analysis of variance (ANOY A) followed by Mann-Whitney V tests for comparisons between groups. Analysis of memory data was performed using ANOY A and post hoc Bonferroni tests. Analysis of histologic data was per­formed using one-way ANOY A followed by the Newman­Keuls test for comparison between groups. Analysis of physi­ologic (temperature, blood pressure) data was performed using a one-way ANOY A followed by post hoc Bonferroni tests.

RESULTS

Study 1

Neurologic motor function. In study I, the composite neuroscores of sham (uninjured) rats treated with rhIL­Ira (high dose) were not statistically different from un­injured animals treated with vehicle at any time point evaluated. Compared with uninjured animals, vehicle­treated, brain-injured animals exhibited significant motor deficits at 48 hours (P < 0.05, Fig. IA) and I week (P <

0.05, Fig. IB) after injury, although some recovery was noted in the brain-injured animals over time. Treatment of brain-injured animals with low-dose rhIL-lra (l0 mgt kg/dose) resulted in a slight, but nonsignificant improve­ment in motor function at 48 hours and 1 week compared with vehicle treatment (Figs. IA and IB). Motor function in brain-injured rats treated with high-dose rhIL- lra (100 mg/kg/dose) was not different at 48 hours (Fig. lA), but the recovery noted in vehicle-treated brain-injured ani­mals was not observed at 1 week in brain-injured animals treated with high-dose rhIL-lra (Fig. IB).

rhlL-I ra TREATMENT IN TBI 1121

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FIG. 1. Composite neuroscore examination at 48 hours (A) and 1 week (8) after fluid-percussion (FP) brain injury in study 1. The graphs indicate individual scores and medians of each group. *,

a significant difference from sham animals; t, significant differ­ence from vehicle-treated brain-injured animals.

Study 2

Cognit ive function. Lateral FP brain injury induced significant cognitive deficits at 42 hours in vehicle­treated animals compared with uninjured animals treated with either vehicle or rhIL- lra (P < 0.05, Fig. 2). Postin­jury administration of rhIL-1 ra significantly improved the cognitive ability of brain-injured animals compared with vehicle-treated injured animals (P < 0.05). Notably, the spatial memory scores of brain-injured animals re­ceiving rhIL-ira were not significantly different from sham-injured animals.

Neurologic motor function. Neurologic motor function of sham-injured rats treated with rhIL- I ra (900 mg/kg) was not statistically different from that of sham-injured animals treated with vehicle at any time point evaluated. Lateral FP brain injury resulted in significant motor defi­cits at 48 hours (P < 0.05, Fig. 3A), I week (P < 0.05, Fig. 3B), and 2 weeks (P < 0.05, data not shown) in both vehicle- and rhIL- Ira-treated rats. Treatment of brain­injured rats with rhIL-ira had no beneficial or detrimen­tal effect on posttraumatic motor function over the 2-week evaluation period.

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FIG. 2. Memory score of animals tested in the Morris water maze at 42 hours in study 2. Graph indicates mean ± SD of the data. *,

a significant difference in mean from sham (uninjured), animals; t, a significant difference from injured, vehicle-treated animals (P < 0.05).

Histologic analysis. Quantitation of neuronal cell loss in the injured cortex demonstrated that high-dose rhIL­I ra was effective in attenuating neuronal loss in area 2 of the parietal cortex (Fig. 4A) and the temporal cortex (Fig. 4B) (P < 0.05 compared with injured, vehicle-

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FIG. 3. Composite neuroscore at 48 hours (A) and 1 week (8) after FP brain injury in study 2. The graphs indicate individual scores and medians of each group. *, a significant difference from sham animals.

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1122 K. L. SANDERSON ET AL.

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FIG. 4. Quantitative neuronal counts in area 2 of the injured parietal cortex (A), temporal cortex (B), area CA3 of the hippocampus (e), and the dentate hilus of the hippocampus (D) after FP brain injury. *, a significant difference from sham animals (P < 0.05); t, sig­nificant difference from brain-injured, vehicle treated rats (P < 0.05).

Vehicle Low Dose High Dose --IL-lra --

Vehicle Low Dose High Dose --IL-lra--

Sham Injured --- Sham Injured ---

treated rats for both regions). Although lateral FP brain injury resulted in neuronal loss in area I of the ipsilateral parietal cortex and the medial geniculate nucleus and ventroposterior lateral thalamus of the ipsilateral thala­mus, neither low- nor high-dose rhTL-} ra was neuropro­tective in these regions (Table i). Neuronal loss was not observed in the ipsilateral lateral septal nucleus, substan­tia nigra, or retrosplenial agranular cortex after brain in­jury (Table }). Treatment with high-dose rhIL-ira after brain injury did not exacerbate neuronal loss in any re­gion examined.

Compared with uninjured rats, brain-injured rats treated with vehicle demonstrated marked neuronal loss in area CA3 (Fig. 4C) and the dentate hilus (Fig. 4D) of the ipsilateral hippocampus at I week after injury. Quan­titative analysis (manual cell counting) revealed signifi-

cant sparing of both CA3 and dentate hilar neurons in brain-injured rats treated with high-dose rhTL-l ra (P <

0.05 compared with brain-injured, vehicle-treated rats) but not in those treated with low-dose rhTL-lra (Figs. 4C and 4D).

Drug and physiologic monitoring. Analysis of the con­tents of the osmotic pumps from study 2 after removal from the animals revealed concentrations of drug re­maining in each pump. Animals treated with rhIL-ira received an average of 45.58 ± 4.14 mg of rhTL-lra over a 5-day period. Concentrations of rhTL-l ra delivered to sham (uninjured) animals (22.93 mg/kg/day) were iden­tical to those administered to injured animals (22.73 mg/ kg/day). Lateral FP brain injury did not affect mean ar­terial pressure, heart rate, or core body and brain tem­perature over the 2-hour postinjury study period (Table

TABLE 1. Regional neuronal counts at one week after lateral fluid-percussion brain injury

Mean neuronal counts

Region Sham Inj-vehicle Inj-Iow dose Inj-high dose

ParCI 155 (± I I ) 30* (± 39) 14* (± 58) 52* (± 58) MGN 4 I (± 7) 9* (± 9) 8* (± 7) 14* (± IS) VPL 36 (± 7) 8* (± 4) 9* (± 7) 11* (± 1l) RSA 68 (± 9) 67 (± 9) 62 (± 12) 65 (± 7) SN 29 (± 3) 29 (± 5) 25 (± 5) 27 (± 5) LSN 72 (± 5) 70 (± 7) 86 (± 8) 69 (± 8)

Rats were subjected to lateral FP brain injury and treated with either vehicle (Inj-veh, n =

15), low-dose rhIL-lra (10 mg/kg, Inj-Iow dose, n = 16), or high-dose rhIL-Ira (100 mg/kg, Inj-high dose, n = 14) as described in Methods. Neuronal counts were determined in area I of the parietal cortex (ParC!), the medial geniculate nucleus (MGN), ventroposterior lateral thalamus (VPL), retrosplenial agranular cortex (RSA), substantia nigra (SN), and lateral septal nuclei (LSN) as described in Methods.

* Significant difference compared to sham (uninjured) rat brain. Values represent mean neuronal counts ± SO in the entire brain region.

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rhlL-lra TREATMENT IN TBI 1123

2). No differences were observed in temporal muscle temperature, core body temperature, mean arterial pres­sure, or heart rate of brain-injured animals treated with 100 mg/kg rhIL-lra or vehicle (Table 2).

DISCUSSION

This study demonstrates that systemic postinjury ad­ministration of high-dose rhIL-lra can significantly at­tenuate cognitive dysfunction and regional neuronal loss associated with experimental brain trauma in rats. The improvement in memory function observed in this study may be related to the ability of rhIL- I ra to inhibit neu­ronal damage in brain regions known to be essential for the retention of a spatial memory task in the rat (Smith et aI., 1991). Cognitive deficits after experimental brain in­jury have been associated with neuronal loss in the hip­pocampus, and treatments that prevent cell loss in the CA3 pyramidal layer of the hippocampus may concur­rently attenuate spatial memory dysfunction (Hicks et aI., 1994; Smith et aI., 1993).

Trauma-induced memory deficits have been shown to correlate with the loss of hilar neurons in rats (Hicks et aI., 1993), and this is the first report of an attenuation of trauma-induced hippocampal hilar cell loss by a pharma­cologic agent. Hilar cells of the dentate gyrus are selec­tively vulneral5le in models of TBI and are thought to be intimately involved in memory function (Hicks et aI., 1993; Lowenstein et aI., 1992). The ability of IL-113 to inhibit long-term potentiation in vitro (Bellinger et aI., 1993) has been related to its ability to induce the gen­eration of reactive oxygen species, which are known to inhibit long -term potentiation (Pellmar et aI., 1991) and to be expressed after TEl (Shohami et aI., 1997). Murray and Lynch (1998) have observed a relation between the upregulation of IL-113 in the dentate gyrus and impaired long-term potentiation, which was associated with the induction of reactive oxygen species. The neuroprotec­tive effects in the dentate gyrus of rhIL-lra in the current study may, therefore, be related to the cognitive im-

provement observed in the rhIL-lra-treated animals. Al­though the protection of cells against trauma-induced injury demonstrates the therapeutic potential of rhIL-I ra treatment, the relation between posttraumatic behavioral dysfunction and neuronal cell loss is not straightforward. Treatment with trophic factors such as NGF or moderate hypothermia have been previously shown to be effica­cious in attenuating cognitive deficits induced by TEl without any apparent protective effects on neurons in the injured hippocampus (Bramlett et aI., 1997; Sinson et aI., 1995). The current study emphasizes the importance of incorporating multiple behavioral and histologic mea­sures to conduct a thorough evaluation of pharmacologic efficacy of novel experimental compounds.

In study 1, vehicle-treated brain-injured animals re­covered somewhat with respect to the neurologic motor scores during the first week after injury. Although brain­injured animals treated with high-dose rhIL-I ra did not demonstrate this improvement, we do not believe that these data imply that this dose of rhIL-lra had a detri­mental effect on motor function. In study 2, the magni­tude of the recovery of vehicle-treated animals was not as great as in study 1, and the comparison between rhIL­I ra-treated and vehicle-treated animals showed no dif­ference in motor function scores. Previous research has shown that rhIL-lra improves motor function after middle cerebral artery occlusion (Garcia et aI., 1995). The composite scores for motor function obtained in study 2 at 7 days after injury in rhIL-1 ra-treated animals were virtually identical with the scores of rhIL-Ira­treated animals in study I, we believe that these data indicate no beneficial or deleterious effect of rhIL- I ra on posttraumatic motor function.

Our histologic observations regarding the attenuation of neuronal cell loss in vulnerable areas of the cortex support those of Toulmond and Rothwell (1995), who previously reported that central (intracerebroventricular) administration of rhIL-lra significantly reduces cortical neuronal cell loss in the brain after FP TBI. Admin-

TABLE 2. Physiological parameters after lateral.fluid-percussion brain injury

Time (min)

Outcome Group Baseline S IS 60 120

MAP (mm Hg) Sham 94 ± S 94 ± 6 92 ± 9 82 ± 7 92 ± 14 Inj-veh 91 ± 18 96 ± 7 88 ± 18 83 ± 20 86 ± 14 Inj-rhIL-I ra 117 ± 12 114 ± 19 114 ± 19 102 ± S 112 ± 6

Core body Sham 37.0 ± O.S 37.4 ± 0.7 37.6 ± O.S 38.0 ± 0 37.7 ± 0.33 temperature Inj-veh 36.8 ± 0.3 36.2 ± 0.9 36.3 ± 0.7 36.9 ± 0.8 37.4 ± 0.8 (0C) Inj-rhIL- I ra 36.S ± 0.8 36.4 ± 0.3 36.S ± 0.3 37.2 ± 0.6 37.3 ± 0.7

Temporalis Sham 3S.1 ± O. I 3S.7 ± 0.3 35.7 ± 0.3 3S.7 ± 0.2 3S.4 ± 0.1

temperature Inj-veh 34.5 ± 1.0 35.0 ± 1.2 3S.0 ± 1.2 35.0 ± 1.0 35.7 ± 0.8

(0C) Inj-rhIL-Ira 35.1 ± 0.5 35.6 ± 0.5 35.4 ± 0.2 35.5 ± 0.3 36.2 ± 0.3

Sham-injured control rats (sham, n = 3) and brain-injured rats treated with vehicle (Inj-veh, n = 4) or high-dose rhlL-lra, (100 mg/kg, Inj-rhIL- I ra, n = 4) were monitored for mean arterial blood pressure (MAP), heart rate, core body temperature, and brain (temporalis muscle) temperature as described in Methods. All values represent group means ± SD.

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1124 K. L. SANDERSON ET AL.

istration of rhIL-lra also has been shown to be neuro­protective after ischemic and excitotoxic damage (Betz et aI., 1995; Garcia et aI., 1995; Loddick and Rothwell, 1996; Relton et aI., 1996), and attenuation of the isch­emic inflammatory response has been reported in mouse brain using an adenoviral vector to induce overexpres­sion of rhIL-lra (Yang et aI., 1995). In addition to pre­vention of cortical cell loss, our observations indicate that rhIL-lra is efficacious in attenuating both cognitive dysfunction and the marked hippocampal cell loss asso­ciated with TBI.

Since the amount of rhIL- I ra administered daily to the injured animals represents a potentially large colloid pro­tein load (almost 2 g/kg over 1 week), this colloid load may have had hemodynamic or local effects on edema (which were not assessed in the current study). Early studies by Albright et ai. (1984) suggest the possibility of important effects of colloid administration after brain injury, whereas more recently, Belayev et ai. (1998) demonstrated a beneficial effect of albumin (3 g/kg) on functional and histologic outcome after lateral FP brain injury in rats. Although no significant hemodynamic changes were observed during the first few hours after brain injury and rhIL-I ra administration, hemodynamic effects may have been observed with a larger number of animals or at later postinjury time points. However, the short plasma and tissue half-life of rhIL-lra (approxi­mately 30 minutes) suggests that prolonged hemody­namic effects would be unlikely.

The mechanisms underlying the apparent neurotoxic effects of IL- l are largely unknown. Inhibition of N­

methyl-o-aspartate receptor-activated brain damage in

vivo by rhIL-lra suggests that IL-l may directly effect or participate in glutamate-mediated excitotoxicity (Rothwell and Relton, 1993). Localized striatal injec­tions of IL- l [3 have been reported to exacerbate ischemic brain damage by specific actions in the striatum, which can influence damage in distal regions of the cortex (Stroemer and Rothwell, 1998). Recent reports illustrat­ing the occurrence of apoptotic cell death after experi­mental TBI (Colicos and Dash, 1996; Conti et aI., 1998; Rink et aI., 1995) raise the possibility that receptor an­tagonism of IL- I may exert an antiapoptotic neuropro­tective effect in the injured brain. Interleukin- I [3 also has been shown to upregulate APP expression in vitro and in

vivo (in nonneuronal cells) and regulate the synthesis of APP mRNA in culture, implicating this cytokine in the pathogenesis of Alzheimer's disease (Sheng et aI., 1996). A marked increase in APP immunoreactivity has been reported after experimental brain trauma (Pierce et aI., 1996). Also, IL-l increases inducible nitric oxide syn­thase mRNA expression and subsequent release of nitric oxide in nonneuronal cells, which has been suggested to contribute to neuronal cell death in the injured brain through apoptotic and/or necrotic mechanisms.

J Cereh Blood Flow Metab. Vol. 19, No. 10, 1999

In contrast to its putative role as a pathologic mediator of cell damage and death after CNS injury, IL- I also may have protective effects in the setting of CNS injury. Up­regulation of IL-I [3 in astrocytes in fetal mice subjected to brain lesions has been associated with a protective reconstruction of a glial limitans, which may be involved with healing and repair (Scripter et aI., 1997). Interleu­kin-l [3 also has been shown to increase levels of NGF twofold to fourfold in brain regions known to have nu­merous IL- I [3 receptors, including the cerebral cortex and hippocampus (Carman-Krzan et aI., 1991). Trans­plantation of fibroblasts transfected with an adenoviral vector expressing rhIL-lra into traumatically injured cor­tex was found to decrease the upregulation of NGF after cortical stab wound- and weight drop-induced trauma (DeKosky et aI., 1996). Since NGF may play a protective role in the traumatically injured brain, the trophic and potentially beneficial actions of endogenous NGF could be compromised after administration of rhIL-lra. These data suggest that although acute treatment of rhIL- I ra has beneficial effects, chronic administration of this drug must be considered with caution because of the potential for prolonged inhibition of NGF in the CNS. Our study is the first to reveal comprehensive behavioral and his­tologic effects of systemic administration of rhIL-l ra in a rodent model of head injury. In view of the apparent neuroprotective effects of rhIL-lra, this drug may have therapeutic potential in treatment of clinical brain injury.

Acknowledgments: The authors thank Seamus Fernandez, Kelli McDermott, and Joshua Leitner for invaluable technical assistance in surgical preparations and behavioral testing, Jeanne Marks for preparation of the manuscript, and Desiree Davis for housekeeping.

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