temporal modulation of cytokine expression following focal cerebral ischemia in mice

Ž .Brain Research 820 1999 45–54

Research report

Temporal modulation of cytokine expression following focal cerebralischemia in mice

Jennifer K. Hill a,) , Lisa Gunion-Rinker c, Doris Kulhanek b, Nikola Lessov c, Songte Kim b,Wayne M. Clark c, Michael P. Dixon c, Rae Nishi b, Mary P. Stenzel-Poore a,1,

Felix P. Eckenstein b,c,1

a Department of Molecular Microbiology and Immunology, Oregon Health Sciences UniÕersity, Mail Code L220, 3181 Sam Jackson Park Road, Portland,OR 97201, USA

b Department of Cell and DeÕelopmental Biology, Oregon Health Sciences UniÕersity, Portland, OR, USAc Department of Neurology, Oregon Health Sciences UniÕersity, Portland, OR, USA

Accepted 20 October 1998

Abstract

There is increasing evidence that the inflammatory response plays an important role in CNS ischemia. The murine model of focalischemia, however, remains incompletely characterized. In this study we examined expression of several cytokines and the vascularadhesion molecule E-selectin, in order to characterize the molecular events following stroke in the C57BLr6J mouse. Using a

Ž .multi-probe RNAse protection assay RPA , mRNA for 19 cytokines was analyzed following permanent and transient occlusion of themiddle cerebral artery in mice. In addition, samples from the same mice were analyzed by reverse transcriptase-polymerase chain reactionŽ .RT-PCR to evaluate E-selectin mRNA expression levels. Several cytokine mRNAs showed a similar expression pattern in bothpermanent and transient CNS ischemia while others showed a temporal expression pattern that was dependent on the type of stroke. For

Ž . Ž . Ž .both models, mRNA levels of TNFa rose early 4 h followed by IL-6 10–18 h and a comparatively late increase 96 h in TGFb1.IL-1a , IL-1b and IL-1ra levels showed a model dependent shift in temporal expression. Reperfusion appeared to delay the induction ofthese cytokines. Temporal changes in cytokine mRNA expression in the mouse CNS occur following ischemic damage. Our findingsdemonstrate the utility and power of multi-probe RPA for evaluation of changes in cytokine mRNA levels. Moreover, this study is, to ourknowledge the first to show temporal changes in cytokine mRNA in mouse cerebral ischemia, forming a basis for further exploration ofthe roles of these cytokines in modulating ischemic neuronal damage in this model. q 1999 Elsevier Science B.V. All rights reserved.

Keywords: Cytokine; Mouse; Cerebral ischemia; E-selectin

1. Introduction

The inflammatory response following cerebral ischemiainvolves the infiltration of peripheral leukocytes from thecirculation into the brain as well as upregulation of pro-in-flammatory cytokines. Expression of the cytokines IL-1b,TNFa and IL-6 peak at 12–24 h following focal ischemiaw x18 . Recent evidence suggests that each of these cytokinesmay lead to exacerbation of neuronal damage followingstroke. Elevated levels of IL-6 in the peripheral circulationof stroke patients correlate with the severity of strokew x w x4,38 and functional recovery of the patient 10 . Baroneet al. recently demonstrated that blocking endogenous

) Corresponding author. Fax: q1-503-494-6862; E-mail:[email protected]

1 Both authors contributed equally to this work.

TNFa significantly reduced infarct size in rats with per-manent or transient middle cerebral artery occlusionŽ . w xMCAo 1 . Finally, a role for IL-1b in the pathogenesisof ischemic brain damage is supported by the increased

Ž .infarction resulting from intracerebroventricular i.c.v in-w xjections of this cytokine in rat brain 43 .

In animal models, adhesion of polymorphonuclearleukocytes to brain capillaries occurs within 6–12 h afterfocal ischemia and is followed by transmigration of leuko-cytes into the brain tissue; this was recently observed in

w xthe mouse model of ischemia 2,3,9,13,14 . E-selectin, anadhesion molecule expressed on activated endothelial cells,has recently been demonstrated to be crucial for the initia-

w xtion of rolling of infiltrating leukocytes 21 . Consistentwith this observation, E-selectin mRNA has been shown toincrease in rat ischemic cortex within 6–12 h and remain

w xelevated for up to 2 days following ischemia 42 . Circulat-

0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.Ž .PII: S0006-8993 98 01140-8

( )J.K. Hill et al.rBrain Research 820 1999 45–5446

ing soluble E-selectin levels were shown to be increased inw xpatients with acute ischemic stroke 17,42 , further impli-

cating a role for brain endothelial E-selectin expression inthe inflammatory events following ischemia.

The rodent is a particularly suitable model of ischemicbrain damage since there are marked similarities between

w xthe cerebral circulation of rodents and humans 16 .Whereas the vast majority of the studies have been per-formed in rats, the recent availability of transgenic micehas led to increased interest in characterizing murine mod-

w xels of stroke 15,24,44 . Mutant mouse models, in somecases representing overexpression of specific moleculesand in others bearing null mutations, have proven to beinvaluable in determining the relative roles of certainmolecules in specific regulatory pathways and diseases.Genetically altered mice offer a unique opportunity toexamine the role of specific gene products. In the contextof cerebral ischemia mice have been generated that lackmany of the hormones or receptors shown to be involved

w xin stroke in several different species 6,25,32,47 . Themouse is able to be manipulated genetically far easier thanany other mammal, thus supporting our attempts and thoseof others to develop this physiologic paradigm in themouse rather than trying to reproduce the genetic systems

w xof the mouse in other species 31 . Two common geneti-cally manipulated strains of mice are C57BLr6 and SV-129. Recently C57BLr6 mice were shown to be moresusceptible to transient forebrain ischemia than SV-129

w xmice 19 . C57BLr6 mice were chosen for this studyw xbecause of their highly reproducible stroke size 44 , sus-

ceptibility to stroke, and popularity as a transgenic back-ground strain.

To define the molecular events following ischemic braindamage in the mouse we used a multi-probe RPAŽ .RiboQuant Kit, PharMingen as a novel method to exam-ine the time course of expression of 19 cytokines follow-ing permanent ischemia and transient ischemia with reper-fusion. Furthermore, to begin to correlate these cytokineswith leukocyte infiltration we examined mRNA regulationof the endothelial adhesion molecule E-selectin, which hasbeen implicated in the extravasation of leukocytes into

w xinflamed tissue 5 and has been shown to be upregulatedw xin the CNS following cerebral ischemia in the rat 42 .

In the present study we have used the novel approach ofa multi-probe RPA to analyze several molecules that havebeen shown by others to be modulated during ischemia in

w xseveral species 1,26,27,38,39,42 . Although the inflamma-tory process has been suggested to play a critical role in

w xreperfusion injury 12 , we have chosen to examine themodulation of several pro-inflammatory cytokines in bothpermanent and transient ischemia to more fully character-ize the ischemic process in the mouse model of stroke.Consistent with previous findings in other species, wefound that TNFa , IL-1b, IL-6, and the adhesion moleculeE-selectin also appear to be modulated following cerebralischemia in the mouse.

2. Materials and methods

2.1. Mice

Ž .C57BLr6J mice male, 6 weeks, 20 g were obtainedfrom Jackson Laboratories and housed in an AALACapproved facility, maintained at 218C and given free accessto food and water. All mouse procedures were approved bythe Department of Animal Care in accordance with IACUCguidelines. Mice were housed individually in opaque cagesin a quiet room 24 h before surgery. All groups except forthe 96 h remained in opaque cages until time of sacrifice.Mice in the 96 h group were individually housed and cageswere wrapped in opaque covers 24 h prior to sacrifice.

Ž .Mice ns8rgroup were divided into the followinggroups: 1 h, 4 h, 10 h, 18 h, 96 h and two control groups,

Ž . Ž .unhandled mice U and mice given anesthesia only 0 h .ŽControl animals were anesthetized for 30 min approxi-

.mate length of surgery and then sacrificed immediately.Mice were subjected to either permanent ischemia or 2 hocclusion followed by reperfusion, except in the case ofthe 1 h timepoint, where only permanent ischemia wasperformed.

2.2. Surgery

Cerebral focal ischemia was induced by MCAo. Anes-Ž .thesia was induced by halothane inhalation 4%rl O and2

maintained with 1.5%rl O . The middle cerebral artery2Ž .MCA was blocked by threading 6-0 monofilament nylonsurgical suture through the external carotid to the internalcarotid and finally blocking its bifurcation into the MCAand anterior cerebral artery. The filament was maintainedintraluminally until the time of sacrifice for the permanentischemia model. In the transient ischemia model, the ani-

Ž .mals were re-anesthetized same conditions as above after

Fig. 1. Correlation between infarct area in 1 mm brain slice from centerof MCA territory and total infarct volume. Data points represent individ-

Ž .ual animals ns8 , solid lines represent first-order linear regression.

( )J.K. Hill et al.rBrain Research 820 1999 45–54 47

Fig. 2. Analysis of RT-PCR linear amplification. L3 and E-selectin wereco-amplified in the same tube to determine the linear range of PCRamplification for semi-quantitative PCR of E-selectin mRNA expressionfollowing stroke. The final protocol employed 25 cycles for L3 and 30cycles for E-selectin amplification.

2 h for removal of the monofilament, thereby restoringblood flow. Rectal temperature was monitored in eachmouse during surgery to prevent artifacts induced by bodytemperature fluctuations. Each mouse was weighed at thetime of surgery and 2 h prior to sacrifice. Although all

Ž .animals lost weight 7–10% there was no correlationbetween weight loss and neurological score or infarct

Ž .volume data not shown .

2.3. Neurological assessment

The animals were initially scored clinically for focalneurological deficits 2 h following surgery to identify

Table 1Correlation of clinical focal score and percent infarction in mice withMCAo

Total Occlu- Reper- Average Hemispheric CorrelationbŽ .time sion fusion clinical infarction % coefficient

a Ž . Ž .time time focal score "S.E.M. rŽ . Ž . Ž .h h "S.E.M.

aa18 h 18 0 11.2"2.2 32.6"7.0 0.87a18 h 2 16 14.4"2.0 47.7"6.3 0.74

96 h 96 0 13.7"2.1 32.6"6.2 0.65aa96 h 2 94 8.3"1.6 32.9"6.4 0.85

a Ž . ŽMice ns8rgroup were scored in each of seven categories bodysymmetry, gait, climbing, circling behavior, forelimb symmetry, compul-

.sory circling, and sensory response by three investigators and the scoresŽ .were averaged see Section 2 . The maximum score is 28. One-way

ANOVA showed no significant effect from time or type of stroke.b Percent Infarction was estimated using a 1 mm coronal slice taken fromthe midsection of the ischemic territory; the infarct area in each of two 50mm sections was multiplied by the section’s thickness and summed, this

Ž .sum was divided by the volume of the non-ischemic hemisphere % .One-way ANOVA showed no significant effect from time or type ofstroke.ap-0.01, aap-0.05, t-test for correlation, testing if the correlation isdifferent from zero.

animals with successful MCAo. Animals showing no signsof focal deficit were removed from the study and replacedto maintain a group size of ns8. The mice were againclinically scored 2 h prior to sacrifice; the 1 h group wasscored at 30 min. The score was based on the physicalappearance and behavior of the mouse. We developed ascale specifically for mice to provide a more preciseassessment of the extent of focal damage that might be

w xexpected in individual animals 11 than was possible withthe Standard 6-Point Scale that is commonly used in ratsw x46 . The mice were scored in each of the following sevencategories: body symmetry, gait, climbing, circling behav-ior, forelimb symmetry, compulsory circling, and sensory

Ž .response. Each mouse was scored from 0 no deficit to 4Ž .severely affected in each category by three trained indi-viduals. The mean of the scores for each animal was usedto assess the correlation of focal neurologic signs withinfarct volume.

2.4. Histology

Animals were sacrificed by cervical dislocation to mini-mize stress. Brains were rapidly removed, placed on a

Ž .tissue slicer Stoelting, Wood Dale, IL and covered with

Fig. 3. Correlation of infarct volume with clinical focal score. Data pointsŽ .represent individual animals ns8 , solid lines represent first-order

linear regression. Bold regression line is for permanent occlusion.


soft agarose. The olfactory bulb and cerebellum wereremoved and discarded. A 1 mm coronal mid-section wasfixed in 10% formalin for histological analysis of infarctvolume with luxol fast blue stain. The frontal 3 mm andcaudal 3.5 mm sections were divided into left and righthemispheres and frozen on powdered dry ice.

2.5. Image analysis

Infarct volume was determined from animals in the 18 hand 96 h groups. Our earliest timepoint for visualization ofthe infarct was 18 h. Using a vibratome, two 50 mmsections from the 1 mm slice were mounted onto slides

Ž .and baked at 378C 6 h followed by 2 h in a chloroformrŽ w x.ethanol 1:1 vrv bath. Slides were then stained with

Ž . Žluxol fast blue 3 h , destained deionized water, 0.05%.lithium carbonate, 50% ethanol , stained with Cresyl violet

Ž .30 min , and dehydrated. Video images of the sectionswere collected by a CCD camera mounted on a stereo-mi-croscope, digitized, and the multiple areas of the infarctand the ipsilateral hemisphere were measured. We havefound a very high correlation between the area of infarct ina slice taken from the center of the MCAo territory and the

Ž 2 .estimated infarct volume Fig. 1, r s0.96 . The volumeof an infarct was estimated using a method similar to that

w xpublished by Swanson et al., 37 , the infarct area in eachŽ .section two sections was multiplied by the section’s

Ž .thickness 50 mm and summed over the two sections. Thepercent of the hemisphere involved can be calculated as:

Ý Infarct Volume rrrrr Normal Hemisphere Volume( ) ( )i i

=100This is a statistical correction for artifactual increases ininfarct size due to tissue edema. Edema can cause anapparent increase in infarct size by increasing the overallsize of the hemisphere. Infarct volume was also calculatedin mm3.

2.6. Cytokine gene expression

Total RNA was prepared from frozen tissue samplesŽ .using Tri-Reagent Molecular Research Center . Individual

hemispheres were homogenized in 5 ml of Tri-Reagent,total RNA was extracted in chloroform, ethanol precipi-tated and, stored at y80 C. Equal amounts of RNA fromeach of the eight individual samples was combined to formpools of RNA for timecourse analysis. Animals from the18 h groups were analyzed individually.

ŽReverse transcriptase-polymerase chain reaction RT-.PCR was used to study the temporal expression of E-

selectin in the ischemic and non-ischemic brain. TotalŽ . Ž . ŽRNA 1 mg was primed with oligo dt 12–18 1 mg;

. yPharmacia and reverse transcribed with RNAse H Su-Ž .perscript II 200 U; GIBCO BRL for 1 h at 378C using

conditions recommended by the manufacturer. The RT

Ž .Fig. 4. TNFa , IL-6, TGFb1 and TGFb2 are modulated during ischemia. Representative multi-probe RPA one of three kits showing cytokine expressionŽ . Ž .for ischemic R and non-ischemic L hemispheres; pooled RNA from eight animals for each lane. L32 and GAPDH are housekeeping RNAs used as

loading controls. ) Indicates lane containing free probe; lines are drawn from free probe to protected band. ¥ Indicates nonspecific probe degradationproduct.


products were stored at y208C. PCR primers used forE-selectin amplification were designed based on similar

Ž Xregions in the rat sequence 5 -CGACATGCACGTTT-X X X.GACTGT-3 and 5 -ATGTGATGATGTCCCTCGGC-3

w x w x42 . L3, a housekeeping gene 33 , was included as aninternal standard for PCR amplification using primers taken

Ž Xfrom the published sequence 5 -TGCCAGGTCATCCG-CATCATTG-3X and 5X-TGACATCAATCATCTCATC-

X. w xCTGCC-3 33 . L3 and E-selectin were co-amplified inthe same tube. Co-amplification of E-selectin and L3

allows for normalization within each tube and controls forvariation between tubes. Test amplifications were run todetermine the linear range of amplification for L3 andE-selectin. Fig. 2 shows that linear amplification for E-selectin lies between 25 and 35 cycles whereas L3 amplifi-cation is linear between 20 and 30 cycles. Thus, to obtainsimilar levels of amplification while remaining within thelinear range of the reaction we chose 25 cycles for L3amplification and 30 cycles for E-selectin. The followingPCR conditions were determined to be optimal for amplifi-

Ž .Fig. 5. Quantitative analysis of cytokine mRNAs following ischemia. Temporal pattern of the relative mRNA levels of the seven of nineteen cytokinesthat were detected and quantitated by multi-probe RPA analysis after permanent and transient MCAo. Data represent ischemic hemispheres only. Relative

ŽRNA levels were determined by PhosphorImage analysis and normalized to L32 expression. Data are from one experiment pooled RNA from eight.animalsrtimepoint .


Žcation: 200 ng cDNA, 2.5 U AmpliTaq polymerase Per-.kin-Elmer Cetus , and in a total volume of 50 ml reaction

Ž .mixture containing 20 mmolrl mM L3 or E-selectinprimers. L3 primers were added to each sample after fivecycles for a total of 30 cycles for E-selectin and 25 cyclesfor L3. The cycling conditions were: 3 min at 968C for 1cycle; 1 min denaturing at 948C; 1 min annealing at 548C;0.5 min extension at 728C for 30 cycles; ending with a 10min extension at 728C. PCR products were electrophoresedthrough 1.5% agarose gel and visualized with ethidiumbromide. A digital photograph was scanned on a ScanJet

Ž .IIc Hewlett Packard and analyzed with NIH Image. ForPCR assays heart RNA from endotoxin treated mice wasused as a positive control for E-selectin expression. Thecorrect product was identified by Southern analysis using aw 32 xg- P ATP end-labeled oligo primer located internal tothose listed above for E-selectin.

Cytokine profiles were determined by RPA. RiboQuantMulti-Probe RNAse Protection Assay is a commercial kitŽ .PharMingen for detecting and quantifying mouse cy-tokine and chemokine mRNA. Templates of distinct size

w32 xwere used to generate a P -labeled anti-sense RNAprobe set and hybridized in excess to target RNA. Briefly,

Ž . Ž .total RNA 10 mg was hybridized 568C; 18 h with thelabeled probe sets, followed by RNAse treatment andanalyses of protected bands on a denaturing 5% polyacryl-amide gel. Gels were subjected to PhosphorImage analysisand bands were quantitated by densitometry. Relative cy-tokine levels were calculated by normalizing the specificcytokine band to the ribosomal RNA L32 band included inthe panel of probes with the kit. A second housekeepingRNA, GAPDH, is included in the multi-probe kits, how-ever it is more highly expressed than L32 and thus difficultto include in quantitative comparisons between levels ofcytokine and GAPDH mRNA expression. The ratio of L32to GAPDH expression was similar with all assays there-fore we chose L32 as our normalization control since wecould detect our target mRNA without saturating the L32signal on the PhosphorImager.

2.7. Statistical analyses

All results are presented as the mean"S.E.M. Statisti-cal comparisons were made using a One-way ANOVAfollowed by Tukey’s test as post-hoc, or by t-test forcorrelation, as appropriate. A two-tailed probability of less

Ž .than 5% p-0.05 was considered statistically signifi-cant.

3. Results

3.1. Neurological deficit and lesion Õolume

The extent of focal damage was initially estimated byneurological evaluation performed on individual animals.

Since neurologic function in the MCAo model is influ-enced by location as well as size of the infarct, weassessed whether the magnitude of the neurologic deficitwas predictive of the infarct volume. Two hours prior to

Ž .sacrifice a clinical score see Section 2 was given to eachanimal from the 18 h and 96 h permanent and transientocclusion groups and infarct volumes were determined at

Ž .the time of sacrifice Table 1 . As shown in Fig. 3 andTable 1, the clinical focal scores correlate with infarctvolume and thus may be a valuable predictor of theseverity of stroke in this animal model.

3.2. Cytokine expression

Brain tissue from ischemic and non-ischemic hemi-spheres was analyzed using three separate commercialRNAse protection kits containing probes for a total of 19different cytokines. Fig. 4 is a representative multi-probeRPA of ischemic and non-ischemic mouse brain using asingle RPA kit that detects eight different cytokine mR-NAs. Seven of the 19 cytokines analyzed were detected bythis method; quantitation by PhosphorImage analysis issummarized in Fig. 5. Several cytokines showed a similartemporal expression pattern in both permanent and tran-sient ischemia; others showed an expression pattern uniqueto the transient or permanent ischemia model. Specifically,we observed a similar temporal pattern in both ischemia

Table 2Focal score vs. relative cytokine levels 18 h following MCAo

a bFocal score Relative levels of cytokine mRNA

IL-1a IL-1ra IL-1b TNFa IL-6

Permanent7.5 qq7.5 qq q q qq q8 q8 qqq q q q8.5 q8.5 q18 qq qq qq qq qq21 qq qq qq qq qqq

Reperfusion9 q9.5 q q q10 q12 q q14 qqq qqq qqq qqq qqq14 qqqq qqqq qqqq qqqq qqqq21 q qq q qq qq25 q q q q

a Ž . ŽClinical focal score scale from 0–28 based on neurological exam see.Section 2 was given to individual animals from the 18 h permanent and

transient occlusion groups.bRelative levels of cytokine mRNA determined by multi-probe RPAŽ . Ž . Ž .RiboQuant . Values ranging from undetectable blank , low q , moder-

Ž . Ž . Ž .ate qq , high qqq , to very high qqqq were determined bynormalization to L32 and PhosphorImage analysis.


paradigms for TNFa , IL-6, and TGFb1: mRNA levels ofŽ . Ž .TNFa rose early 4 h followed by IL-6 10–18 h and a

Ž .comparatively late increase 96 h in TGFb1. In the caseof TGFb2, mRNA levels showed very little change inresponse to ischemia. TGFb1 levels increased late in bothmodels which may be due to activation of neuronal repairprocesses in the penumbra, an area of the infarct whereneurons survive longer, consistent with the neuroprotective

w xrole ascribed to this growth factor 29 . In contrast, thepattern of temporal regulation of the cytokines IL-1a ,

Ž .IL-1b, and IL-1 receptor antagonist IL-1ra differed be-tween the transient and permanent occlusion models: in-

Ž .duction of these cytokines peaked earlier 10 h in thepermanent occlusion model compared with 18 h in thetransient occlusion model. Low levels of a few of the

Ž .cytokines were detected in the contralateral non-ischemicŽ .hemisphere data not shown . The following cytokines

were analyzed, however, no mRNA was detected in eitherŽthe controls or after MCAo 48 h exposure to autoradiog-

.raphy film showed no detectable mRNA : IL-2, IL-4, IL-5,Ž .IL-9, IL-10, IL-12 both p35 and p40 subunits , IL-13,

IL-15, interferon-g, interferon-b, lymphotoxin b, and tu-mor necrosis factor b.

To determine whether the levels of expressed cytokinewere associated with the severity of clinical deficit individ-

Žual animals were examined 18 h after MCAo permanent.and reperfusion . Table 2 shows the focal clinical score of

each animal compared to expression levels of a subset ofŽ .cytokines IL-1a , IL-1ra, IL-1b, TNFa and IL-6 . Inter-

estingly, increased deficits as measured by the clinicalscore are more frequently associated with increased IL-6

Fig. 6. E-selectin mRNA is modulated during cerebral ischemia. RelativeE-selectin mRNA levels quantitated by RT-PCR analysis of ischemichemispheres after permanent or transient MCAo. The PCR-amplifiedDNA bands of E-selectin were normalized to L3 that was co-amplified in

Žthe same tube. Data are from one stroke experiment pooled RNA from.eight animalsrtimepoint . cDNA and subsequent PCR was generated

independently three times for each group. Data are presented as theaverage of three amplifications"S.E.M. Permanent and reperfusion werestatistically analyzed separately, ))) p-0.001 compared to all time-points, One-way ANOVA, Tukey’s post-hoc.

expression. Six of the eight mice with clinical scores of 10or greater expressed IL-6, whereas only one of the remain-ing eight with scores less than 10 expressed IL-6, howeverat a very low level. Although this is a small number ofanimals, this trend suggests further investigation into thecorrelation of cytokine expression and neurological out-come.

3.3. Modulation of E-selectin mRNA

E-selectin mRNA expression has been shown to in-crease during ischemia in rat and non-human primatesw x23,42 . RT-PCR analysis of the ischemic hemispheres ofmice undergoing permanent occlusion and ischemia withreperfusion showed marked increases in the expression

Ž .levels of E-selectin Fig. 6 . Expression levels of E-selec-Ž .tin peaked slightly earlier 10 h in animals undergoing

Ž .permanent ischemia than in the transient model ;18 h .

4. Discussion

It is increasingly clear that a reliable mouse model isneeded to elucidate the specific mechanisms leading toneuronal damage during stroke. We have begun to assessthe nature and temporal pattern of specific molecular

w xevents associated with stroke 1,26,27,38,39,41,42 to clar-ify the mechanisms of ischemic brain injury in the mouse.In past studies, a Standard 6-Point scale has been used todetermine the neurologic deficit following ischemia in ratw x46 . We have developed a 28-Point Focal scale to evaluate

w xsimilar changes in mice 11 . Although the Standard 6-Pointscale can be adapted for use with mice, in our hands itlacks the sensitivity to distinguish between mildly andmoderately impaired animals. The 28-point scale providesa greater range and finer resolution of neurological deficitsfacilitating the characterization of behavior exhibited byeven mildly impaired animals. The present study demon-strates that the 28-Point Focal score correlates well with

Žthe infarct volume determined by the amount of neuronal.damage and therefore indicates that the neurologic deficit

determined by the focal score may be a useful predictor ofthe severity of the stroke in the mouse.

Several of the cytokines analyzed here have been re-ported to be modulated in the rat model of cerebral is-

w xchemia 27,39,41 ; however, this is the first evaluation ofchanges in cytokine expression in the mouse brain follow-ing MCAo. Recent observations using a mouse model ofMCAo implicate a number of cytokines and growth factorsin the modulation of neuronal damage following cerebral

w xischemia 6,22,30,34,45 . Although it is not yet clearwhether there is a direct correlation between altered cy-tokine expression and neuronal damage, several recentstudies support a strong association between individual

w xcytokines and neurological outcome 1,4,36 . In particular,


TGFb1 appears to play a neuroprotective role in ischemiasince smaller infarcts were observed in mice injected i.c.v.

w xwith TGFb1 34 . In addition, TGFb1 has been shown tow xupregulate ciliary neuronal trophic factor 29 and may be

w xupregulated in the ischemic penumbra 26 . Our resultsŽ .showing modulation of TGFb1 late 96 h in ischemia are

consistent with a view that this mediator may be involvedwith neuronal repair processes.

Although a direct role for IL-1 and the endogenous IL-1Ž .antagonist IL-1ra in modulating cerebral ischemia in the

mouse has not been established several studies suggest thatthe presence of IL-1 may exacerbate ischemic damage. Weshow an early upregulation of IL-1b and IL-1ra, in addi-tion, we demonstrate a modest increase of IL-1a levelsfollowing both transient and permanent ischemia. More-over, it has been shown by others that overexpression of

w xIL-1ra attenuates ischemic brain injury in mice 45 , sug-gesting an important role for the IL-1 system in cerebralischemia. This is further supported by the finding that miceexpressing a mutated gene for IL-1b converting enzymeŽ .thereby lacking active IL-1a and IL-1b also showed

w xdecreases in brain injury following ischemia 22 .In our studies we show an early and robust increase in

TNFa mRNA levels, however the role of TNFa in is-w xchemic brain injury is unclear 35 . Recently, it was shown

that blocking endogenous TNFa significantly reduced in-farct size in rats with permanent or transient MCAo,indicating that endogenous TNFa may exacerbate neu-

w xronal damage 1 . In contrast, a recent report by Bruce etw x Ž .al. 6 indicates that brain injury ischemic and excitotoxic

Žwas worse in mice lacking receptors for TNFa p55 and.p75 double knockout mice suggesting that TNFa may

w xserve a neuroprotective function 6 . While this finding inTNFa receptor knockout mice implies a neuroprotectiverole for TNFa , it should be pointed out that there may bea ’compensatory increase in IL-1 in these mice which

w xcould exacerbate neuronal damage in ischemia 35 . It iswell known that TNFa exerts cytotoxic actions on non-neuronal cells and in the case of neurons, it can be either

w x w xneurotrophic 8 or neurotoxic 7,20 depending on theexperimental conditions. There is recent evidence thatadministration of TNFa directly into the brain can cause

w xboth protection from 30 or exacerbation of ischemic braininjury, which may depend on timing of administration anddose of TNFa .

IL-1b, IL-6, and TNFa are not detectable in the unin-jured mouse CNS. A marked induction in expression ofmRNA of these cytokines is seen after CNS injury. Datafrom the present study indicate that expression of cy-tokines may be regulated in an hierarchical manner in theCNS after stroke. Fig. 7 is a model of the temporalrelationship of IL-1b, TNFa , IL-6 and E-selectin in thepermanent and reperfusion models of MCAo. These curveswere generated based on the results shown in this study;however, they are theoretical models of the kinetics ofgene expression and are not based on interpolated data.

Fig. 7. Schematic representation of mRNA kinetics during MCAo. Theo-retical model of gene expression representing the temporal relationship of

Ž .IL-1b, IL-6, TNFa and E-selectin during transient A and permanentŽ .B MCAo in mouse.

TNFa mRNA is detected early in permanent ischemia,prior to increased levels of E-selectin that are seen first at

Ž .4 h following permanent occlusion Fig. 7B . Early in-creases in TNFa mRNA levels are also detected in tran-sient ischemia, followed by increased IL-1b mRNA levelsŽ .Fig. 7A . E-selectin mRNA levels in transient ischemia,however, in contrast to permanent ischemia, increase laterbeginning at ;10 h and peaking at ;18 h. Takentogether our data suggest there is a temporal relationshipamong these mediators of inflammatory damage; TNFa

appears to be the primary inducer of IL-1b and E-selectin.IL-1b levels rise at approximately the same time as, orshortly after, E-selectin mRNA levels following permanent


ischemia, suggesting that IL-1b may not act as the primaryinducer of E-selectin. This does not rule out a role forIL-1b in maintaining E-selectin levels in ischemic damage.

Consistent with published data in the rat and humanw x1,38 , TNFa and IL-6 expression are associated withgreater neurologic deficits and larger infarcts. It is interest-

Žing to note that a large infarct indicated by a focal scoreŽ ..of 25 Table 2 shows little activation of any cytokine.

w xKrupinski et al., 26 made similar observations whenlooking at TGFb1 expression in human stroke patients,suggesting that severe damage may impair a cell’s abilityto efficiently respond and induce de novo mRNA synthe-sis. Table 2 suggests that there may be some threshold ofcytokine expression given that in the 18 h reperfusion

Ž .group where the largest infarcts were observed Table 1there is significant cytokine expression in mice with aneurological score of 14, however there is very littlemRNA in a mouse with a focal score of 12. The signifi-cance of the mRNA expression for these cytokines de-scribed in the present study must be interpreted withcaution, since no evidence has been provided for an in-crease in the corresponding protein levels.

To further understand the significance of the roles ofTNFa , IL-1, IL-1ra, IL-6, and TGFb it will be importantto identify the cells that produce and respond to thesecytokines and growth factors. Early cellular responses toischemic damage in the brain are likely to involve reactivemicroglia at the site of damage. Activated microglia canrelease many cytotoxins such as nitric oxide, excitatoryamino acids and the inflammatory cytokines IL-1b, TNFa ,and IL-6. Reactive astrocytes remain in the middle ofcontroversy regarding their role in exacerbating neuronaldamage. It is generally believed that astrocytes are apositive influence since they produce neuroprotectivemolecules such as nerve growth factor, ciliary neurotrophic

w xfactor, and basic fibroblast growth factor 28 .In addition to activation of resident cells, acute inflam-

mation in response to focal ischemia in the brain is charac-terized by infiltration of polymorphonuclear leukocytesfrom the circulation. The infiltrate is largely comprised of

w xneutrophils 2,3,9 , particularly during the initial stages ofw xinflammation 9 . Recent observations utilizing a mouse

model of cerebral ischemia have shown increasedmyeloperoxidase activity 24 h following stroke, reflecting

w xinfiltrating neutrophils 3 . Consistent with this observa-tion, increased gene expression of cell adhesion moleculesICAM and E-selectin has been observed in ischemic brainw x14,40,42 . In our studies using the mouse model of MCAopeak E-selectin mRNA levels were observed after 10 h in

Ž .both permanent and transient focal ischemia Fig. 4 ,consistent with the time course of cellular infiltration

w xobserved in other animal models of stroke 2,9 .We have demonstrated that mRNA levels for several

important molecules of the inflammatory cascade are mod-ulated following cerebral ischemia in the mouse. Some ofthe cytokines described here have already been genetically

w xmutated in mice 25,32,47 , facilitating future dissection ofthe roles of these cytokines in modulating neuronal dam-age in the ischemic CNS.

Acknowledgements

The authors would like to thank Dr. William Woodwardand Alex Ho for expert technical assistance. We gratefullyacknowledge M. Brown, S. Stevens, K. Heldwein, J. Auldand Drs. T. O’Hare, G. Wiens, E. Whitcomb and M.Rittenberg for critical review of the manuscript. This workwas funded by Program Project grant NS17493, National

Ž . Ž .AHA Grant-in-Aid M.S.-P. , NS25767 R.N. and a grantŽ .from NIA AG07424 F.E. .

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temporal modulation of cytokine expression following focal cerebral ischemia in mice

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