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Neurobiology of Disease 6, 200–208 (1999)

rain IL-1 b Increases Neutrophil and Decreasesymphocyte Counts through Stimulation ofeuroimmune Pathways

hristian Woiciechowsky, Britta Schoning, Nadja Daberkow,atrin Asche, Wolfgang R. Lanksch, Wolf-Dietrich Docke,*nd Hans-Dieter Volk*epartment of Neurosurgery, Charite-Campus Virchow-Klinikum, Humboldt-University oferlin, Augustenburger Platz 1, D-13353 Berlin; and *Institute of Medical Immunology,harite-Campus Mitte, Humboldt-University of Berlin, D-10098 Berlin, Germany

eceived November 16, 1998; revised January 22, 1999; accepted for publication March 1, 1999

Leukocytosis after cerebral injury is well described and may participate in the generation of cerebraldamage. However, the mechanisms of brain-induced leukocytosis are still speculative. Since it isknown that proinflammatory cytokines are involved in neuroimmunomodulation and since others andwe have demonstrated high cytokine levels in the cerebrospinal fluid following injury, we supposedthat brain cytokines may also influence leukocyte counts. In order to evaluate this hypothesis, weestablished an animal model using continuous intracerebroventricular (icv), intrahypothalamic (ih), orintravenous infusion of the proinflammatory cytokines tumor necrosis factor (TNF)- a and IL-1 b.Controls received vehicle solution. With this experimental paradigm we could show that icv and ihinfusion of IL-1 b but not TNF- a dramatically increased neutrophil counts, whereas lymphocytesdropped. Blocking the hypothalamic–pituitary–adrenal (HPA) axis by hypophysectomy abolished theneutrophilia, whereas the lymphopenia remained unchanged. Furthermore, application of the b2-adrenoreceptor antagonist propranolol prevented the decrease of lymphocytes and diminished the

Article ID nbdi.1999.0242, available online at http://www.idealibrary.com on

neutrophilia. All parameters normalized within 48 h after termination of infusion. So, our resultsdemonstrate that brain IL-1 b can modify blood leukocyte counts through stimulation of both the

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sympathetic nervous system (SNS) and the HPA axis

NTRODUCTION

Recent experimental evidence confirms the interrela-ionship between the central nervous, the neuroendo-rine, and the immune system. Indeed, extensiveuality exists in the use of neurotransmitters, cyto-ines, hormones, and receptors each system displaysKeller et al., 1981; Reichlin, 1993). Cytokines are mostmportant mediators of immune cells which can acti-ate central nervous system (CNS) pathways like theypothalamic–pituitary–adrenal (HPA) axis and theympathetic nervous system (SNS) (Payne & Krueger,992; Rivier et al., 1989; Sapolsky et al., 1987; Sundar et al.,
990; Watkins, 1994; Weiss et al., 1994; Woiciechowsky et al.,998). The subsequent release of cortisol and catechol-mines can modulate immune cell functions and gener-
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te changes in circulating immune cell counts (Chel-icka Schorr et al., 1992; Friedman & Irwin, 1995;apcala et al., 1995; Mastorakos et al., 1995; Meisel et al.,996; Platzer et al., 1995; Sundar et al., 1989; van deroll et al., 1996). In this case catecholamines releaseeutrophil stores, and corticosteroids cause a decrease

n the egress of neutrophils from circulation (Boggs,967).Furthermore, various kinds of brain injury like

rauma, ischemia, or subarachnoid hemorrhage aressociated with leukocytosis (Keskil et al., 1994; Neil &ruickshank, 1974; Parkinson & Stephensen, 1984). Itas been suggested that increased cell counts reflect a
tress situation with increased catecholamine and cor-icosteroid activity in addition to tissue necrosis. Addi-ionally, urinary catecholamine and plasma cortisol
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evels were correlated with white blood cell counts andpositive relationship was demonstrated (Cruicks-

ank et al., 1970). However, the pathways of sympa-hetic and neuroendocrine stimulation after cerebralnjury, ischemia, or subarachnoid hemorrhage are stillpeculative and stress or increased intracranial pres-ure cannot be unique mechanisms.

Interestingly, others and we have shown high concen-rations of proinflammatory cytokines, e.g., interleukinIL)-1b, IL-6, and tumor necrosis factor (TNF)-a in theerebrospinal fluid (CSF) after brain injury, ischemia,nd subarachnoid hemorrhage (Beamer et al., 1995;euerstein et al., 1994; Holmin et al., 1997; Mathiesen etl., 1993, 1997; Woiciechowsky et al., 1997). Further-ore, we demonstrated a correlation between release

f proinflammatory cytokines into the CSF, activationf the HPA axis and the SNS, and systemic immunode-ression in neurosurgical patients (Asadullah et al.,996, 1995; Woiciechowsky et al., 1998). Immunodepres-ion was reflected by the down-regulation of mono-ytic human leukocyte antigen-DR expression and exivo TNF-a secretion capacity (Asadullah et al., 1996;oiciechowsky et al., 1998). With this background we

peculated that proinflammatory brain cytokines arelso involved in the brain-mediated leukocytosis withredominantly high neutrophil counts. Furthermore,e considered that activation of the HPA axis as well

s the SNS by brain cytokines could be key mecha-isms for the induction of immune effects following

njury or ischemia.In order to confirm our hypothesis, an animal modelas established using continuous cytokine infusion

nto the brain for 2 days. To address the significance ofhe brain region and the specificity of the mediators forhe systemic immune effects two different rat recombi-ant (rr) cytokines (IL-1b and TNF-a) were infused

nto two different compartments, the cerebroventricu-ar system (icv) and the hypothalamus (ih). The hypo-halamus is the supposed sphere of action for brain-IL-b. However, most of the IL-1 receptors in the brain areocalized in the dentate gyrus and choroid plexusRothwell, 1991). Therefore, interbrain connections maylso be involved in the brain IL-1b-induced effects.ontrol animals received vehicle solution. Further-ore, in order to demonstrate that the CNS is the site

f action for the infused cytokines, additional controlseceived iv infusion of vehicle solution, rrIL-1b, andrTNF-a. In this experimental paradigm we monitoredhe changes in total and differential (neutrophils and

rain IL-1b Induces Neutrophilia and Lymphopenia

ymphocytes) leukocyte counts in an acute (after 4 h ofytokine infusion) and a chronic (after 48 h) stage. Inrder to evaluate the importance of the HPA axis and

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he SNS for the immune response, supplementaryxperiments were performed with hypophysecto-ized rats and blocking b2-adrenoreceptors with pro-

ranolol. Finally, to analyze the recovery of the immu-ological changes, investigations were extended to 48after termination of the IL-1b infusion in one group.

ATERIAL AND METHODS

nimals

Approval for the study was achieved from thenimal Protection Board of the Senat of Berlin. A totalf 130 male Sprague–Dawley rats (Harlan Winkel-ann, Borchen, Germany) weighing 250–350 g at the

ime of surgery were used. The animals were dividednto 13 groups of 10 rats each. Three different solu-ions, vehicle (4% human serum albumin dissolved inhysiological saline (DRK-Blutspendedienst, Springe,ermany), rrIL-1b (R&D Systems, Minneapolis, MN),

nd rrTNF-a dissolved in vehicle solution (Laboserv,taufenberg, Germany), were administered in threeifferent locations: icv, ih, and intravenous (iv). For theontinuous application of the cytokines, osmoticinipumps (96-h duration, 100µl, 1µl/h; Model 1003D;lza Corp., Palo Alto, CA) were used. The concentra-

ion of cytokines in pumps was 1µg dissolved in 100µl% HSA. The delivery rate was 10 ng cytokine in 1 h.his dosage was calculated according to previouslinical and animal studies demonstrating high levelsf proinflammatory cytokines in the brain after injuryr meningitis and adapted to studies showing effectiveentral IL-1 doses for alterations of immune cell func-ions (Beamer et al., 1995; Feuerstein et al., 1994;

olmin et al., 1997; Mathiesen et al., 1993, 1997; Sharieft al., 1992; Woiciechowsky et al., 1997). According tohe specifications of the suppliers, the endotoxin con-amination was negligible (,0.1 ng/mg cytokine) asetermined by the LAL method. The length of the

nfusion was 48 h. In order to guarantee an immediatenfusion after implantation, minipumps were preincu-ated for 16 h at 37°C prior to surgery according to theanufacturer’s instructions. In order to investigate theechanisms of how icv infusion of IL-1b can induce

ystemic immune effects, additional experiments wereerformed with hypophysectomized rats. Hypophysec-

omy (HPX) was performed by the animal supplierweek prior to operative procedures by a para-

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haryngeal approach as described by Waynforth (1980),nd rats treated with the b2-adrenoreceptor antagon-st propranolol ((S)-(2)-propranolol hydrochloride;

Copyright r 1999 by Academic PressAll rights of reproduction in any form reserved.

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igma-Aldrich, Deisenhofen, Germany). Propranololas injected intraperitoneally (10 mg/kg) before and 12,

4, and 36 h after initiation of cytokine infusion. Theosage and injection intervals were calculated accord-

ng to previous descriptions of effective SNS-blockingchemes in rat (Cignarelli et al., 1990). In order to studyhe recovery of the immunological changes, the experi-

ent was extended to 48 h after termination of theL-1b infusion (96 h in total) in one group of animals.

mplantation of Minipumps for Cytokine Infusion

The implantation of ALZET pumps into the brainas performed as follows: rats were anesthetizedsing a gas mixture of isoflurane (1.0% v/v) and2O/O2 (50.0/25.0%, v/v), and an L-shaped cannula

f the ALZET brain infusion kit was placed into the leftentricle or the left anterior hypothalamus. The coordi-ates were 0.8 mm posterior and 1.2 mm lateral toregma and 3.8 mm below the dura for the ventriclend 1.8 mm posterior and 0.7 mm lateral to bregmand 9.0 mm below the dura for the anterior hypothala-us according to Paxinos and Watson (1982). The

annula was fixed with two stainless steel screws andounted to the skull with dental cement. The screws

cted as an anchor to secure both the external portionf the cannula and the entire implantation site. Theinipumps were implanted subcutaneously in the

ape of the neck and attached to the cannula via aubing. For the iv infusion a PE-10 tube was asepticallylaced into the left femoral vein to the level of the venaava inferior and connected to the pump. The pumpas also implanted subcutaneously.

xperimental Procedures

Forty-eight or 96 h after initiation of infusion, animalsere euthanized. Afterward, brains were excised and

rozen in liquid nitrogen and stored at 280°C until usedor histological examinations. In order to confirm theorrect localization of the cannula, cryostat sections (10µm)f each brain at the level of the cannula were mountednto a glass slide, dried, and stained with H&E andnalyzed at the light microscopic level and comparedith the anatomic atlas (Paxinos & Watson, 1982).

lood Samples

02

Heparin-anticoagulated venous blood samples (50µlach) from the ophthalmic venous plexus were col-

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opyright r 1999 by Academic Pressll rights of reproduction in any form reserved.

ected from rats 1 day before surgery, 4 and 48 h afterurgery, and, in one group, additionally 48 h afterermination of cytokine infusion using the methodescribed by Reilly (1995).

ounting and Differentiation of White Blood Cells

At each time point, the number of leukocytes wasetermined. Therefore, two samples, 10µl each, of thelood probes were diluted 1:20 in 3% acetic acid andounted twice in a Neubauer chamber, so that theverage value for each blood sample resulted from aourfold counting. Furthermore, blood smears wereerformed for leukocyte differentiation. The bloodells were stained with May–Grunwald and 2 3 100ells were counted in two different regions. The pres-nce of neutrophils, lymphocytes, and monocytes wasetected by their characteristic morphology. The val-es were calculated as absolute neutrophil and lympho-yte counts per milliliter of blood volume. In order tovercome preoperative differences in leukocyte countsnd to make the values comparable between theifferent groups the data are expressed as percentagesf the preoperative (0-h) value. Only in the 96-h study,

n which values were compared within one animalroup, were the absolute numbers used.

tatistical Analysis

Data are presented as means 6 SEM. Statisticalnalyses included the Kruskal–Wallis analysis of vari-nce (ANOVA) for independent samples. If this testemonstrated significant intergroup differences Mann–hitney U tests were performed for the comparison of

tudy groups. A Friedman repeated measures ANOVAn ranks was used for dependent samples. If this testas positive the Wilcoxon test was used for paired

amples in follow up. A P lower than 5% was consid-red significant. For multiple comparisons the a prob-bility was adjusted according to Bonferroni.

ESULTS

istological Verification of the Cannula Position

In all animals with implanted brain cannulas the

Woiciechowsky et al.

orrect position of the cannula was verified histologi-ally. Only data from animals with correct cannulalacement and without injury to the surrounding brain

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at least 9 from 10 animals in each group) werevaluated (Fig. 1).

rain-IL-1 b Induces Peripheral Neutrophilia butlso Lymphopenia

The preoperative absolute numbers of leukocytesaried between all animals from 0.54 to 2.9 3 106/mlor neutrophils and from 5.6 to 11.1 3 106/ml forymphocytes. There were also small intergroup differ-nces in the mean values (Fig. 3). Therefore, fortatistical analyses the percentage of the preoperative0-h) value was used. Only in the 96-h study in whichalues were compared within one animal group werehe absolute numbers used.

IG. 1. Representative coronal section of a rat brain 48 h afternitiation of icv infusion of rrIL-1b (H&E staining). Arrowheads


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oint at the cannula duct, demonstrating the correct position of theannula tip in the ventricle without any damage to the surroundingrain tissue.

In all animals, including vehicle-treated rats, a tem-orary increase of neutrophils and decrease of lympho-ytes were observed 4 h after surgery (Fig. 2). How-ver, icv (by ANOVA, for neutrophilia after 48 h P 5.0004, for lymphopenia after 4 h P 5 0.003 and after 48P 5 0.006) and ih (by ANOVA, for neutrophilia after

8 h P 5 0.0009) infusion of IL-1b induced moreronounced and long-lasting (for 48 h) changes of

eukocyte counts (Fig. 2). Similar but less profoundffects were observed in animals receiving iv infusionf IL-1b (by ANOVA, for neutrophilia after 48 h P 5.002, for lymphopenia after 4 h P 5 0.0006, Fig. 2).nterestingly, interruption of the HPA axis by HPXbolished the neutrophilia (by ANOVA, P 5 0.004),hereas the lymphopenia remained unchanged (Fig.

). On the other hand, blocking the SNS transmissionith the b2-adrenoreceptor antagonist propranolol led

o a fast recovery (after 48 h) from the IL-1b-inducedcute lymphopenia (by ANOVA, P 5 0.002) andiminished the neutrophilia (Fig. 3).

he Brain IL-1 b-Induced Peripheral Neutrophiliand Lymphopenia Were Reversible 48 h afterermination of Cytokine Infusion

The absolute numbers of neutrophils and lympho-ytes also showed an increase during the icv infusionf IL-1b. Forty-eight hours after termination of theytokine infusion neutrophil and lymphocyte countsormalized, demonstrating that the observed systemic

mmune effects were directly affected by the cerebralytokine application (Friedman’s test, for neutrophils5 0.0003 and for lymphocytes P 5 0.001, Fig. 4).

ISCUSSION

Many studies have demonstrated leukocytosis fol-owing brain injury, ischemia, or subarachnoid hemor-hage (Keskil et al., 1994; Neil & Cruickshank, 1974;arkinson & Stephensen, 1984; Spallone et al., 1987). Itas suggested that the increased cell counts reflect a

tress situation with activation of the HPA axis and theNS in addition to tissue necrosis. So, a direct relation-hip between elevated plasma cortisol and urine cat-cholamine levels and leukocyte counts was demon-trated (Neil et al., 1974). However, the mechanisms ofctivation of the neuroimmune pathways after brain

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njury are speculative and stress cannot only be respon-ible for the described effects. Additionally, there areeports documenting normal leukocyte counts on ad-


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IG. 2. Changes in neutrophil and lymphocyte counts during continuous infusion of IL-1b, TNF-a, or vehicle solution into theerebroventricular system (i.c.v.), the anterior hypothalamus (i.h.), or the blood (i.v.). Blood samples were obtained at different time points and

04 Woiciechowsky et al.

ells were differentiated and counted after staining under light microscope. Data are expressed as percentages of total neutrophils and lymphocytes ofhe pretreatment (0-h) value 6 SEM. ***P , 0.001, **P , 0.01, *P , 0.05 versus vehicle application at the same time point, Mann–Whitney U test.


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ission after subarachnoid hemorrhage (at the timeoint of maximal environmental stress) and increasedumbers at the time of clinical deterioration (Spallonet al., 1987).

Furthermore, after various kinds of brain injuryroinflammatory cytokines (e.g., IL-1b, IL-6, andNF-a) are released into the brain and the CSF (Feuer-tein et al., 1994; Holmin et al., 1997; Mathiesen et al.,

IG. 3. Changes in neutrophil and lymphocyte counts during contr treatment with the b2-adrenoreceptor antagonist propranolol (10 mells were differentiated and counted after staining under light mymphocytes of the pretreatment (0-h) value 6 SEM. ***P , 0.001,

ann–Whitney U test. 11P , 0.01, versus IL-1b application alone at


993, 1997; Woiciechowsky et al., 1997). Moreover,rain cytokines can alert blood immune cell functionsy activating the HPA axis and the SNS (Payne &

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rueger, 1992; Rivier et al., 1989; Sapolsky et al., 1987;undar et al., 1990; Watkins, 1994; Weiss et al., 1994;oiciechowsky et al., 1998). Corresponding to these

ata we supposed that brain cytokines are also respon-ible for the shift of leukocyte counts after brain injury.

Our results show early (4 h after initiation of infu-ion) immune effects in all groups, including vehicle-nfused rats, with a slight neutrophilia and lymphope-

icv infusion of vehicle solution, IL-1b, or IL-1b and additional HPXvery 12 h). Blood samples were obtained at different time points andope. Data are expressed as percentages of total neutrophils and0.01, *P , 0.05 versus vehicle application at the same time point,e time point, Mann–Whitney U test.

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ia. In our opinion, this demonstrates a stress-inducedmmune response resulting from surgery. However,hese changes normalized within 48 h in vehicle and


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NF-a-infused rats, whereas IL-1b-infused rats showedmore severe lymphopenia after 4 h and the immune

lterations were present during the whole infusioneriod. Comparing the different infused compart-ents, icv cytokine application was most effective in

nducing neutrophilia and lymphopenia. This is inter-sting considering the fact that the hypothalamus ishe supposed side of action for brain IL-1b. However,s already mentioned, brain IL-1 receptors are local-zed predominantly in the dentate gyrus and choroidlexus and it is conceivable that icv infusion is moreffective to stimulate cells in these regions, which alsoave neuronal connections to the hypothalamus andrain stem (Rothwell, 1991). There is also one othereport documenting leukocytosis after injection ofL-1b into the hypothalamus. However, the authorsid not describe a mechanism of the brain IL-1b-ediated leukocytosis (Morimoto et al., 1988). Further-ore, Robertson et al. (1988) demonstrated that a-mela-

ocyte-stimulating hormone is capable of inhibitinghe ability of peripherally administered IL-1 to induceeutrophilia, suggesting the central action of thisytokine (Robertson et al., 1988). Our results also showhat iv application of IL-1b produces a long-lastingeutrophilia, whereas lymphopenia was visible onlyfter 4 h of cytokine infusion. We suggest that stimula-ion of central immune pathways by peripheral IL-1b

IG. 4. Changes in neutrophil and lymphocyte counts during and aere obtained at different time points and cells were differentiated an

umbers 3 106/ml blood 6 SEM. ###P , 0.001, ##P , 0.01 versus 0-h

06

s responsible for these effects. The stronger immunehanges after icv infusion reflect the fact that brain IL-1irectly affects neuroimmune pathways, whereas IL-1

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pplication into blood results first in a cytokine dilu-ion and then the diluted cytokine has to reach therain (Kent et al., 1994).Since in clinical and experimental studies others ande have demonstrated that brain-mediated immune

ffects are associated with sympathetic activation asell as with stimulation of the HPA axis by proinflam-atory cytokines (Asadullah et al., 1995, 1996; Sundar

t al., 1990; Watkins, 1994; Woiciechowsky et al., 1996,998), we suggested that these neuroimmune path-ays are also involved in the brain IL-1b-mediatedodification of leukocyte counts.In order to evaluate this hypothesis, additional

xperiments with animals receiving icv infusion ofL-1b were performed by turning off the HPA axishrough HPX and blocking sympathetic b2-adrenore-eptors with propranolol.

The data show that HPX abolished the neutrophilia,hereas the lymphopenia remained unchanged. On

he other hand, propranolol prevented the long-lastingymphopenia (lymphocyte counts recovered within 48

of cytokine infusion), whereas the neutrophilia wasiminished. These results confirmed the involvementf the HPA axis and the SNS in the brain IL-1b-riggered immune effects. Furthermore, it seems thatytokine infusion into the CSF results in a more diffuse

mination of continuous icv infusion of IL-1b for 48 h. Blood samplested after staining under light microscope. Data are expressed as cellilcoxon test for paired samples in follow-up.


istribution than the local application into the hypo-halamus. In this way an additional stimulation ofympathetic neurons could contribute to the more

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evere immune consequences compared with the ihroup.Interestingly, lymphocytes dropped rapidly with

owest values after 4 h, whereas neutrophils achievedighest numbers after 48 h. It seems that action ofatecholamines on lymphocytes with reduction ofroliferation and changes in migration is responsible

or these early effects (Madden et al., 1995). On thether hand the increase of neutrophil counts seems torise predominantly from action of corticosteroids,hich decrease the egress of neutrophils from circula-

ion. This may take more time to produce measurablehanges (Boggs, 1967). In the beginning we also countedeukocytes after 24 h and values were comparable tohe numbers after 48 h (data not shown). Therefore, iteems that neutrophils reach maximum numbers be-ween 4 and 24 h. Marimoto et al. (1988) also observedeukocytosis induced by microinjection of IL-1 into theypothalamus with maximum levels after 8 h (Mori-oto et al., 1988).Moreover, stopping IL-1b infusion ended in normal-

zation of all peripheral leukocyte counts within 48 h,emonstrating that all effects directly depend on the

nfusion of IL-1b into the CSF.In conclusion, our findings show that icv and ih

nfusion of IL-1b at the dosage of 10 ng/h for 48 hauses an impressive neutrophilia and a severe lympho-enia. Activation of both the HPA axis and the SNS byrain cytokines is distinctly involved in the immuneffects described.

These results may be interesting regarding the ef-ects of leukocytes on brain–blood flow after injury.eukocytosis can contribute to cerebral damage since

eukocytes are less deformable than erythrocytes andherefore a greater pressure gradient is required toorce them through the capillaries, whose diameter

ay be smaller than that of the leukocytes. Underonditions of reduced perfusion pressure, leukocytesay come in contact with the endothelium and may

ot be dislodged even after perfusion pressure iseturned to normal (Dutka et al., 1989; Hallenbeck et al.,986; Heinel et al., 1994; Spallone et al., 1987; Ya-akawa et al., 1987). This mechanical occlusion of the

apillaries may become even more pronounced as aesult of the expression of a number of cell adhesion

olecules (e.g., ICAM, VCAM) (Harlan, 1985). Further-ore, in animal ischemia models a protective effect of

eutrophil depletion was demonstrated (Dutka et al.,989; Hallenbeck et al., 1986; Heinel et al., 1994).


herefore, a reduction of leukocyte counts by blockinghe sympathetic transmission may be an interestingttempt to prevent the second injury syndrome follow-

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ng cerebral injury or ischemia. The sympathetic block-de may also be beneficial in order to prevent brainnjury-induced immunodepression since the SNS-riggered systemic IL-10 release is a key mediator inhis process (Woiciechowsky et al., 1998). Furthernvestigations should evaluate this concept.

CKNOWLEDGMENT

This work was supported by a grant from the Deutsche Forsch-ngsgemeinschaft, SFB 507/C5.

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