Ž .Brain Research Protocols 4 1999 217–222www.elsevier.comrlocaterbres

Protocol

Continuous infusion of proinflammatory cytokines into the brain to studybrain cytokine induced local and systemic immune effects

Britta Schoning a, Peter Elepfandt a, Wolfgang R. Lanksch a, Hans-Dieter Volk b,¨Christian Woiciechowsky a,)

a Department of Neurosurgery, Charite-Campus Virchow-Klinikum, Humboldt UniÕersity, Augustenburger Platz 1, D-13444 Berlin, Germany´b Institute of Medical Immunology, Charite-Campus Mitte, Humboldt UniÕersity, D-10098 Berlin, Germany´

Accepted 6 April 1999

Abstract

Ž .Proinflammatory cytokines are produced in the brain after various kinds of insult ischemia, trauma, infection . In this processŽ . Ž .interleukin IL -1b, IL-6 and tumor necrosis factor TNF -a are most important. These cytokines are key mediators of inflammation.

Ž .Furthermore, these cytokines can act as neurotransmitters and develop direct effects on the central nervous system CNS including fever,sleep and stimulation of the neuroendocrine as well as sympathetic nervous system. Moreover, IL-1b and TNF-a may also be involved inbrain repair and regenerating processes. However, most of the data about the role of cytokines in the brain have been obtained from eitherin vitro studies or bolus injections into the brain parenchyma or cerebroventricular system. On the other hand, it is known that cytokinesare released continuously into the brain after a cerebral insult over a period of 24 to 48 h. In order to further complete the knowledgeabout the interactions between neural and immune cells to overcome the primary insult and initiate repair and regeneration in the CNS, anew animal model of local inflammation reaction was established using chronic intracerebral infusion of rat recombinant cytokines.q 1999 Elsevier Science B.V. All rights reserved.

Themes: Endocrine and autonomic regulation

Topics: Neural-immune interactions

Keywords: MonocyterMacrophage; Neutrophil; Neuroimmunology; Cytokine; Interleukin-1b; Tumor necrosis factor-a; Inflammation; Migration

1. Type of research

The present model is suitable for any study involvingŽthe interactions between cytokines and brain tissue astro-

.cytes, microglia and neural cells . The continuous cytokineinfusion better represents the conditions following a cere-bral insult than bolus injections, since cytokine concentra-tions are stable in the brain parenchyma or cerebrospinal

Ž .fluid CSF over a certain period. In detail, this in vivomodel can be used to study brain-cytokine induced cell

Ž .invasion into the central nervous system CNS , activationof microglia and astrocytes, expression and production ofother mediators, stimulation of neuro-immune pathwayslike the sympathetic nervous system and hypothalamic–

w xpituitary–adrenal axis, etc. 20 .

) Corresponding author. Fax: q49-30-45060977; E-mail:christian.woiciechowsky@charite.de

2. Time required

Ø Fill-up of the osmotic mini-pump: 5 min.Ø Pre-incubation of the pump for 14 h.Ø Surgical preparation of the rat for pump implantation:

15 min.Ø Stereotactically guided placement of the cannula and

implantation of the pump: 15 min.Ø Sacrifice animals, CSF sampling and brain excision: 15

min.

3. Materials

ŽØ Male Sprague–Dawley rats Harlan Winkelmann,.Borchen, Germany weighing 250–350 g at the time of

surgeryŽ .Ø ALZET mini-pumps Model 1003D and ALZET brain

Ž .infusion kit Alza, Palo Alto, CA, USA

1385-299Xr99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.Ž .PII: S1385-299X 99 00022-7

( )B. Schoning et al.rBrain Research Protocols 4 1999 217–222¨218

Ž . Ž .Fig. 1. Left: Surgical exposure of the rat skull with bore-holes for the cannula 1 and the screws 2 . Right: Placement of the cannula of the brain infusionkit connected to the pump via a catheter. The pump is located in the nape of the neck.

ŽØ Stereotactic device for small animals David Kopf In-.struments, Tujunga, CA, USA

w xØ Stereotactic atlas 8ŽØ Drill Dremel Moto-Tool, Stoelting, Wood Dale, IL,.USA

ŽØ Stainless screws SSMS 1r8, 3.2 mm, Bilany Consul-.tants, Dusseldorf, Germany¨

ŽØ Dental cement Harvard Cement, Richter and Hoff-.mann, Berlin, Germany

Ž . Ž . ŽØ Rat recombinant rr interleukin IL -1b R&D sys-.tems, Minneapolis, USA and tumor necrosis factor

Ž . Ž .TNF -a Laboserv, Staufenberg, Germany

4. Detailed procedure

4.1. Animals

Approval for the study was achieved from the AnimalProtection Board of the Senat of Berlin. Male Sprague–

Ž .Dawley rats Harlan Winkelmann, Borchen, Germanyweighing 250–350 g at the time of surgery were used. Theanimals were divided into six groups. Three different

Ž .compounds vehicle, rrIL-1b and rrTNF-a were adminis-tered into two different locations: intracerebroventricularŽ . Ž .i.c.v. and intrahypothalamic i.h. .

4.2. Fill up of mini-pumps

For the continuous application of the cytokines, osmoticŽmini-pumps 96-h duration, 100 ml, 1 mlrh; Model 1003D

.Alza, Palo Alto, CA, USA were used. The concentrationof cytokines in pumps was 10 mgrml, so the delivery ratewas 10 ng cytokinerh. The length of the infusion was 48h. In order to guarantee an immediate infusion of cytokinesafter implantation, mini-pumps were filled and pre-in-cubated in 0.9% saline for 14 h at q378C prior to surgeryaccording to the manufacturer’s instructions.

The three different infusion compounds were: vehicleŽ4% HSA dissolved in 0.9% saline, DRK-Bluts-

. Žpendedienst, Springe, Germany , rrIL-1b R&D systems,. ŽMinneapolis, USA and rrTNF-a Laboserv, Staufenberg,

Fig. 2. Representative histological section of a rat brain before watercooling was used during drilling. Arrows show the zone of heat inducednecrosis on the cortex.

( )B. Schoning et al.rBrain Research Protocols 4 1999 217–222¨ 219

Fig. 3. Representative whole brain photograph directly after excision.Through improvement of the experimental setup the implantation of thecannulas and the fixation screws became safe without any injury to thebrain tissue.

.Germany dissolved in vehicle solution. Endotoxin levelswere below 0.1 ngr1 mg cytokine as determined by thesupplier via the LAL method.

4.3. Implantation of mini-pumps for cytokine infusion

The implantation of ALZET-pumps into the brain wasperformed as follows: rats were anaesthetised using a gas

Ž . Žmixture of isoflurane 2.0% vrv and N 0r0 0.5 lrmin,2 2.0.25 lrmin . An L-shaped cannula of the ALZET brainŽ .infusion kit Alza, Palo Alto, CA, USA was introduced

into the left ventricle or the left anterior hypothalamus.The coordinates were: 0.8 mm posterior and 1.2 mm

lateral to bregma and 3.8 mm below the dura for theventricle and 1.8 mm posterior, 0.7 mm lateral to bregmaand 9.0 mm below the dura for the anterior hypothalamus

w xaccording to Paxinos and Watson 8 . For the placement ofthe cannula and for the screws three bore holes were

Ž .drilled into the skull Fig. 1 . In order to avoid heat relateddamage to the cortex a water-cooling is indispensable

Ž .during drilling Fig. 2 . After insertion, the cannula wasfixed with two stainless steel screws mounted to the skullwith dental cement. The screws acted as an anchor tosecure both the external portion of the cannula and theentire implantation site. The mini-pumps were implantedsubcutaneously in the nape of the neck and attached to thecannula via a tubing.

4.4. CSF sampling and brain excision

Forty-eight hours after initiation of infusion, animalswere sacrificed. Thereafter, the CSF was obtained througha needle puncture of the cisterna magna. The CSF samples

Ž .were objected to cell count Neubauer chamber and mea-surement of cytokine concentrations. Afterwards, brainswere excised, frozen in 2-methylbutane immersed in liquidnitrogen and stored at y808C until sectioning.

4.5. Histological and immunohistological studies

In order to confirm the correct placement of the cannulaand to demonstrate signs of meningoencephalitis cryostate

Ž .sections 10 mm of each brain at the level of the cannulawere mounted onto a glass slide, dried and stained withhematoxylin and eosin, analysed at the light microscopic

w xlevel and compared with the anatomic atlas 8 .The immunohistological staining was accomplished ac-

cording to the ABC-method or the APAAP-method asŽdescribed by the supplier both reagents from Dako,

.Glostrup, Denmark . For labeling of activated microgliaŽthe biotinylated lectin GSA I-B4 Lectin I-B4 from Griffo-

nia simplicifolia, Vector, Linaris, Wertheim-Bettingen,.Germany diluted to 20 mgrml was used followed by

ABC-method. Binding of the monoclonal antibody OX-6

Table 1TNF-a , IL-1b and cell counts in CSF of rats 48 h after i.c.v. or i.h. infusion of vehicle solution, TNF-a and IL-1b

Ž . Ž .Infusion side Solution TNF-a pgrml IL-1b pgrml Cell counts5Ž .10 rml

i.c.v. vehicle -0.7 13.6"5.6 0.2"0.1TNF-a 171.1"45.0 107.6"72.8 3.3"0.1IL-1b -0.7 1556.0"187.5 312.0"90.4

i.h. vehicle -0.7 23.8"9.1 0.0TNF-a 24.2"13.0 11.5"5.1 5.0"0.6IL-1b -0.7 88.2"25.1 43.6"3.7

TNF-a and IL-1b levels were determined by ELISA after aspiration of the CSF through a needle puncture of the cisterna magna. Cell counts weredetermined in a Neubauer counting chamber. The data are expressed as mean"S.E.M. pgrml. -0.7: TNF-a concentration was below the detection levelof the ELISA used.

( )B. Schoning et al.rBrain Research Protocols 4 1999 217–222¨220

Fig. 4. Representative CSF smear after staining with Giemsa. The cells inŽ . Ž .the CSF were predominantly neutrophils 1 . However monocytes 2 and

Ž .lymphocytes 3 could also be seen in lower numbers.

Ž .Serotec, Oxford, England , diluted 1:100, specific for ratI-A antigen present on monocytesrmacrophages was de-tected with the APAAP-method.

4.6. Cytokine leÕels in CSF

Cytokine levels in CSF were determined using thefollowing commercially available ELISA kits: ultra-sensi-

Žtive rat TNF-a detection level: 0.7 pgrml, Cytoscreen

Ž .Fig. 5. Coronal section of a rat brain interhemispheric fissure 48 h afterŽ .initiation of i.c.v. infusion of rrIL-1b H&E staining . An impressive cellŽ .invasion into the subarachnoid space interhemispheric fissure and brain

parenchyma with perivascular localisation of cell infiltrates is visibleŽ .arrows .

.USe, Laboserv, Giessen, Germany and IL-1b-ELISAŽdetection level: 3 pgrml, Cytoscreen rIL-1b, Biosource,

.Nivelles, Belgium .

5. Results

The drilling as well as placement of cannula and screwscan be performed without any visible damage to the brain

Ž .tissue Fig. 3 . This is very important since the local andsystemic immune effects studied should directly depend onthe cytokine infusion and not be influenced by the proce-dure itself.

Our results demonstrate that the paradigm described isappropriate to deliver cytokines into the brain. Analysis ofTNF-a and IL-1b concentrations in the CSF revealedhigher levels after i.c.v. infusion compared with infusion

Ž . Ž .into the brain parenchyma i.h. Table 1 . TNF-a wasfound only when rrTNF-a was infused whereas IL-1b waspresent in small amounts even after infusion of vehicle

Ž .solution Table 1 . However, a control group of animalstreated only with narcosis and blood sampling also showed

Ž .low CSF IL-1b-concentrations 7.8"4.8 pgrml . Thismay reflect a stress induced production of IL-1b in thebrain in low amounts due to anaesthesia.

With regard to the cytokine applied, infusion of rrIL-1b

induced a severe meningo-encephalitis with high cell num-Ž .bers in the CSF Table 1 whereas rrTNF-a was less

w xeffective at the same dose 20 . Infusion of vehicle solutiondid not lead to any cell infiltration. The infiltrating cellswere predominantly neutrophils, but monocytes and lym-

Ž .phocytes were also detectable Fig. 4 .The histological and immunohistological examination

revealed a generalised meningo-encephalits after i.c.v. in-Ž .fusion of rrIL-1b Fig. 5 . On the other hand, i.h. infusion

produced an encephalitis focused to the infused site and aconcomitant meningitis was present unilateral and more

Ž .severe at the basis of the forebrain Fig. 6 . Forty-eight

Ž .Fig. 6. Coronal section of a rat brain septum region 48 h after initiationŽ .of i.h. infusion of rrIL-1b H&E staining . Cell invasion is focused to the

Ž .infused site around the cannula duct 1 with perivascular cell infiltratesŽ .2 .

( )B. Schoning et al.rBrain Research Protocols 4 1999 217–222¨ 221

Ž .Fig. 7. Coronal section of a rat brain region of cannula duct 48 h afterinitiation of i.c.v. infusion of vehicle solution. Immunohistochemicalstaining with antibody OX-6 recognizing monocytesrmacrophages. There

Ž .are only few positive cells around the cannula duct arrows .

hours after the beginning of i.c.v. or i.h. infusion ofvehicle, the brain sections showed the response to a smallsterile stab wound trauma. Only little signs of inflamma-tion restricted to the near surrounding of the cannula ductwith microglia activation and OX-6 positive cells, express-

Ž .ing rat antigen I-A, were visible Fig. 7 .Staining of microglia with the lectin GSA I-B4 identi-

fied a high number of positive cells at the cannula duct andin the broader surrounding both for i.c.v. and i.h. applica-tion of TNF-a and IL-1b but only little staining wasvisible after vehicle infusion.

Corresponding to the local effects systemic immuneeffects could also be observed. So, we demonstrated abrain-IL-1b induced switch of immune cells into an anti-

Žinflammatory state low TNF-a and high IL-10 production.after endotoxin stimulation in whole blood cultures . In

addition, animals receiving IL-1b showed obvious signs ofillness including weight loss, with i.c.v. infusion beingmore stressful. In contrast, after administration of TNF-aanimals resembled the vehicle-groups, which seemed nearly

w xunstressed 20 .

6. Discussion

ŽMany effects after a cerebral insult trauma, ischemia,.infection are associated with cytokine production in the

Ž . Žbrain by immune microglia and non-immune cells astro-.cytes, endothelial cells . The stimulus for this cytokine

release is predominantly hypoxia and necrotic cell mate-rial, which induce IL-1b, IL-6 and TNF-a synthesis.These cytokines can generate brain edema through capillarleakage, cell invasion, stimulation of the expression of celladhesion molecules, and support the second injury cascadew x1,5–7,10,13,16,21,22 . Furthermore, these cytokines arealso involved in brain repair and regenerating processesand sometimes it is difficult to distinguish between the

w xpositive and negative effects of cytokines in the brain 12 .

Additionally, behind the proinflammatory cytokines thesame insults also induce the production of anti-inflamma-

Ž .tory cytokines like IL-10 and IL-1 receptor antagonist raw x4,11,18 .

Several in vivo studies have been performed to deter-mine the importance of cytokines in various cerebral pro-

w xcesses predominantly using bolus injections 1,9,10 . How-ever, it is known that cytokine release into the brain after acerebral insult is more continuous about a period of 24 to

w x48 h 4,6,15,18 . In order to further complete the knowl-edge about the interactions between neural and immunecells to overcome the primary insult and initiate repair andregeneration in the CNS, a new animal model of localinflammation reaction was established using chronic in-tracerebral infusion of rat recombinant cytokines.

With this model we were able to show that cytokineapplication into the brain can be performed safely andreliably over 48 h and that the measured cytokine concen-trations in the CSF are comparable to cytokine levels after

w xbrain injury or meningitis 14,16,18 . Histological andimmunohistological studies revealed only a small number

Žof activated microglia and OX-6 positive cells macro-.phages after vehicle infusion. Therefore, the histological

findings and CSF analysis of vehicle infused animalsresembled the normal reaction of brain tissue to a smallsterile stab wound trauma.

Furthermore, it could be demonstrated that IL-1b in thebrain is most potent to induce inflammation and cellinvasion. Additionally, continuous i.c.v. IL-1b infusioninduces systemic immunodepression with monocytic deac-

w xtivation 20 . This corresponds to our clinical studies,where we could demonstrate that cerebral inflammation

w xresults in systemic immunodepression 2,3,18,19 . More-over, this paradigm creates ideal conditions to investigatethe mechanisms of cytokine induced cell invasion into theCNS as well as principles of neuroimmuno-modulation.Furthermore, selective blocking of different pathways ofthe brain-IL-1b mediated local and systemic immune con-sequences could improve our understanding of interactionsbetween immune and neural cells after a defined cerebralinsult in order to restore homeostasis.

6.1. Troubleshooting

In order to guarantee that all local and systemic effectsdepend directly on the brain cytokine infusion it is neces-sary to avoid any damage to the brain tissue due to theprocedure itself. Under these circumstances heat resulting

Ž .from drilling can produce necrosis on the cortex Fig. 2 .Therefore, water cooling is indispensable during drilling.Furthermore, dura should not be injured during drilling andscrews should not be placed to deep to avoid any pressureon the cortex.

6.2. AlternatiÕe and support protocols

To our knowledge there is no comparable method ofcytokine induced cerebral inflammation. Nevertheless,

( )B. Schoning et al.rBrain Research Protocols 4 1999 217–222¨222

there are reports of bolus cytokine application into thebrain with resulting signs of meningitis. However, thecytokine concentrations used in bolus injections are very

w xhigh and do not represent real in vivo conditions 9,10 .

7. Quick procedure

Ž .A Fill-up of the osmotic mini-pumps one day beforeimplantation.

Ž .B Incubation of the pumps for 14 h in order toguarantee an immediate cytokine release after implanta-tion.

Ž .C Next day the implantation of ALZET-pumps intoŽthe brain as follows: preparation of the rat placement of

the rat into the stereotactic device, exposure of the skull,drilling bore-holes—do not forget water-cooling, place-

.ment of the screws . Stereotactically guided introduction ofthe cannula, implantation of the pump in the nape of theneck, fixation of the entire brain infusion kit with dentalcement, closure of the incision.

Ž .D Sacrifice animals after a defined period of cytokineŽ .infusion e.g., 48 h , CSF sampling due to puncture of the

cisterna magna and brain excision.

8. Essential literature references

w xEssential Refs. 1,3,9,10,13,17,18,20 .

Acknowledgements

This work was supported by a grant from the DeutscheForschungsgemeinschaft, Wo 621r2-1 and SFB 507rC5

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