Abstract The effect of transient focal cerebral ischemia onprotein regulation was studied in mice using multipara-metric immunohistochemistry. Injury was characterizedby measurements of blood flow, regional protein synthesisand terminal transferase biotinylated-dUTP nick end la-beling (TUNEL). The proteins studied were selected froma previously established list of differentially regulatedproteins and included the GTPases dynamin, RhoB, CASand Ran BP-1, the transcription factors Nurr1 and p-Stat 6,the protein kinase MAPK p49, the splicing factors SRPK1and hPrp16, the cell cycle control proteins cyclin B1 andNek2, the inflammatory proteins FKBP12 and Rag2, thecell adhesion protein paxillin and the folding proteinTCP-1. Regulation patterns were diverse and comprisedipsi- and/or contralateral up- and down-regulation with orwithout topical association to impeding cell death. Someproteins (SRPK1, TCP-1 and Nurr1) also exhibited post-ischemic translocation from the nucleus to the cytosol. Ourobservations stress the importance of regional analysis forthe interpretation of proteomic data, and contribute to theidentification of new pathways that may be involved inthe evolution of post-ischemic brain injury.

Keywords Immunohistochemistry · Protein expression ·Gene regulation · Focal cerebral ischemia · Translocation

Introduction

Gene profiling methods are increasingly used for the de-tection of disease-relevant alterations of gene expressionin neurological disorders, such as Alzheimer’s disease[16], Parkinson’s disease [11], aging [27], seizures [9, 40],

spinal cord injury [5, 38], traumatic brain injury [29, 30]and cerebral ischemia [23, 25, 28, 40, 45]. The identifica-tion of novel modulators of neuronal death is thought tocontribute to the understanding of injury mechanisms andthe identification of targets for possible therapeutical in-terventions.

As regards brain ischemia, gene profiling by cDNAmicroarrays [25, 36] or serial analysis of gene expression(SAGE) [44] suggest that the number of genes that are up-or down-regulated is high. Even if only those genes areconsidered that are regulated by a factor of more than 10,close to 7% of the total genome is involved [43]. How-ever, this does not mean that these changes are of rele-vance for the evolution of the disease process. Differentialgene profiling is usually carried out by screening mRNAexpression, but as brain ischemia is associated with awidespread and long-lasting inhibition of protein synthe-sis at the translational level, genes that code for short-livedproteins may be superinduced due to the release of feed-back control [2, 8].

Many genes also respond to peri-infarct depolarizations,which spread over the entire hemisphere and which leadto the up-regulation of immediate-early genes in the in-tact, non-ischemic tissue [24]. Finally, the breakdown of theenergy state in the core of the ischemic territory results ininhibition of both transcription and translation, which maybe misinterpreted as active down-regulation. A more reli-able approach for the evaluation of the role of selectivegene regulation for injury evolution is, therefore, the analy-sis of protein translation in combination with independentmarkers of tissue injury.

Recently, we performed the first proteomic investiga-tion of focal brain ischemia, using multi-Western blotanalysis of hemispheric protein extracts [42]. Out of 400proteins with a wide range of regulatory functions, up to45% were up- or down-regulated at various time pointsafter ischemia by a factor of 1.5 or more in both the ipsi-lateral and the contralateral hemisphere. To study the re-gional distribution of some of these proteins, we repeatedthe same experiment, and performed immunohistochem-istry in combination with terminal transferase-biotinylated

Franciska Erdö · Thorsten Trapp · Günter Mies ·Konstantin-A. Hossmann

Immunohistochemical analysis of protein expression after middle cerebral artery occlusion in mice

Acta Neuropathol (2004) 107 : 127–136DOI 10.1007/s00401-003-0789-8

Received: 3 September 2003 / Accepted: 30 October 2003 / Published online: 26 November 2003

REGULAR PAPER

F. Erdö · T. Trapp · G. Mies · K.-A. Hossmann (✉)Max Planck Institute for Neurological Research, Department of Experimental Neurology, Gleueler Strasse 50, 50931 Cologne, GermanyTel.: +49-221-4726210, Fax: +49-221-4726325,e-mail: hossmann@mpin-koeln.mpg.de

© Springer-Verlag 2003

dUTP nick-end labeling (TUNEL) and autoradiographicimaging of protein biosynthesis. This combination allowsthe precise topical association of the regulated proteinswith the ischemic impact, which, in turn, can be differen-tiated into reversible and irreversible injury.

Our data revealed complex expression patterns, whichfrequently expanded beyond the ischemically damagedtissue and which have to be interpreted with care to avoidmisconceptions on the pathogenetic role of regulated pro-teins for the evolution of tissue injury.

Materials and methods

Experiments were carried out according to the National Institutesof Health Guidelines for the Care and Use of Laboratory Animalsand approved by the local authorities. Animals were kept understandard diurnal lighting conditions and had free access to foodand water until the day of the experiment. Anesthesia was inducedby 1.5% halothane and maintained with 0.9% halothane in 70%N2O and 30% O2.

Induction of focal cerebral ischemia

Adult male C57BL/6J mice (Charles River, Germany) weighing22–26 g were used. Three groups of animals (n=4 per group) weresubjected to focal cerebral ischemia for 60 min, followed by 0, 3 and12 h recirculation, respectively. Four animals were used as sham-operated controls.

Focal cerebral ischemia was produced by occluding the middlecerebral artery (MCA) with an intraluminal filament [14]. Duringthe experiments blood flow was measured by laser Doppler flowme-try (LDF) using a 0.5-mm fiberoptic probe (Perimed, Stockholm,Sweden), which was attached to the intact skull overlaying theMCA territory (2 mm posterior, 6 mm laterally from bregma). LDFchanges were monitored throughout the 1-h ischemia and up to 30 min after the onset of reperfusion. A midline neck incision wasmade and the left common and external carotid arteries were iso-lated and ligated. A microvascular clip (FE691; Aesculap, Tuttlin-gen, Germany) was temporarily placed on the internal carotidartery. An 8-0 nylon monofilament (Ethilon; Ethicon, Norderstedt,Germany) coated with silicon resin (Xantopren; Heraus Kulzer,Dormagen, Germany) was introduced through a small incision intothe common carotid artery and advanced 9 mm distal to the carotidbifurcation for occlusion of the MCA. The size of the thread(0.15–0.20 mm) was selected to match the body weight of the ani-mals. One hour after the occlusion the thread was withdrawn to al-low reperfusion of the MCA territory. Sham-operation was per-formed by inserting the thread into the common carotid artery, butwithout advancing it to the MCA.

Forty-five minutes before the animals were killed, L-[4,5-3H]leu-cine (300 µCi/animal, specific activity 151 Ci/mmol; Amersham,Braunschweig, Germany) was administered intraperitoneally toevaluate cerebral protein synthesis (CPS) rates. After the prede-termined reperfusion periods, experiments were terminated underhalothane anesthesia by decapitation. The brains were removed,frozen with liquid nitrogen [32] and serially cut into 20-µm-thickcoronal cryostat sections. Sections were mounted on poly-L-lysine-coated glass slides for immunohistochemistry, terminal transferasebiotinylated-dUTP nick end labeling (TUNEL) and CPS autoradi-ography.

Temperature control

During surgery and up to 30 min after MCA occlusion, rectal tem-perature was kept at 37.0°C using a heating lamp and a heating padconnected to a thermistor. After recovery from the anesthesia, ani-

mals were maintained in an air-conditioned room at approximately22°C.

Immunohistochemistry

Air-dried brain sections were fixed in ethanol/acetone (1:1 v/v) for10 min. Thereafter, the sections were washed for 5 min in phos-phate-buffered saline (PBS), treated with 0.3% hydrogen peroxidein methanol for 20 min to block endogenous peroxidase activity,and given two more 5-min rinses with PBS. For immunostaining,sections were incubated in PBS containing 5% normal goat serumor normal horse serum and 0.3% Triton X-100 for 30 min at roomtemperature. The slides were then incubated for 24 h at 4°C withthe following primary antibodies: CAS goat polyclonal, cyclin B1rabbit polyclonal, dynamin rabbit polyclonal, FKBP12 goat poly-clonal, HIF-1α rabbit polyclonal, Nek2 goat polyclonal, Nurr1rabbit polyclonal, p-Stat6 goat polyclonal, Rag2 rabbit polyclonal,Ran BP-1 goat polyclonal, RhoB rabbit polyclonal, SRPK1 goatpolyclonal and TCP-1α goat polyclonal (all Santa Cruz Biotech-nology), and with hPrp16 mouse monoclonal, MAPK p49 mousemonoclonal and paxillin mouse monoclonal (all BD TransductionLaboratories). Antibodies were diluted 1:100 in PBS containing5% goat serum or normal horse serum and 0.3% Triton X-100, ex-cept RhoB antibody which was diluted 1:200. Thereafter, sectionswere washed three times in PBS and incubated with biotinylatedsecondary antibody (1:100; goat anti-rabbit antibody, goat anti-mouse antibody DAKO Diagnostics, Glostrup, Denmark, or horseanti-goat antibody Vector Co., Burlingame, CA) in PBS contain-ing 5% normal goat serum or normal horse serum and 0.3% TritonX-100 at room temperature for 2 h. After washing in PBS threetimes for 5 min, slices were incubated with streptavidin-horserad-ish peroxidase (Vectastain Elite ABC; Vector) in PBS for 30 minat room temperature. Immunoreactivity was visualized via the de-tection of biotin-streptavidin-peroxidase complex by incubationwith diaminobenzidine tetrahydrochloride (Sigma FAST DAB).Sections were dehydrated with increasing concentrations of ethanoland embedded. The specificity of the immunoreactivity was testedin selected experiments with the immunogene blocking peptide(Santa Cruz Biotechnology) before being used for immunohisto-chemistry according to the instructions of the manufacturer.

TUNEL staining

TUNEL was performed as described previously [47], with minormodifications. Briefly, coronal brain sections were fixed for 15 minin ice-cold 4% paraformaldehyde/PBS, pH 7.4. Subsequently, thesections were washed twice in 70% ethanol (1 min), once in PBS(3 min), and once in 0.3% hydrogen peroxide/PBS (5 min). Afterequilibration for 15 min in TDT buffer (100 mM potassium ca-codylate, 2 mM cobalt chloride, 0.2 mM dithiothreitol), the bufferwas quantitatively removed, sections were incubated in 50 mlTDT-Mix [10 pM biotin-16-dUTP (Boehringer, Mannheim, Ger-many) and 150 U/ml terminal deoxynucleotidyl transferase (LifeTechnologies, Eggenstein, Germany)] in TDT buffer, and coveredwith coverslips. After incubation for 60 min at 37°C, the reactionwas terminated by washing the sections for 15 min in TB buffer(300 mM sodium chloride, 30 mM sodium citrate). Incorporatedbiotin was visualized using the avidin-biotin-peroxidase complexmethod (Vector), as recommended by the supplier. Finally, thesections were dehydrated and embedded in Eukitt (Kindler,Freiburg, Germany).

Measurement of cerebral protein synthesis

For CPS measurement, brain slices were incubated in 10% trichloro-acetic acid to remove labeled free leucine and metabolites otherthan proteins. Subsequently, slices were exposed for 21 days with3H standards to tritium-sensitive x-ray film (Hyperfilm 3H; Amers-ham) for autoradiography of 3H-labeled proteins [32].

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Incidence maps of regional alterations

To evaluate the regional reproducibility of immunostainings forTUNEL and CPS, regional incidence maps were constructed [14].The areas of biochemical disturbances were outlined on represen-tative coronal brain sections from each individual animal and su-perimposed at the level of caudate-putamen and in some cases alsoat the level of the hippocampus. Using the NIH image analysissoftware, the incidence of metabolic alterations was calculated foreach pixel and expressed as a percentage of the number of animalsper group.

Results

General physiological observations

The effects of intraluminal MCA thread occlusion on gen-eral physiological parameters have been described previ-ously [16]. In this study the reduction of regional cerebralblood flow was measured by LDF. For the histochemicalinvestigations, brains were selected according to the fol-lowing criteria: (1) after MCA occlusion the LDF de-clined to below 35% of the basic level, (2) after with-drawal of the thread the LDF returned to the control level(100%) within 15 min. Fig. 1 shows the mean ischemicand post-ischemic LDF values in the three groups submit-ted to 60-min ischemia and 0, 3 and 12 h reperfusion, re-spectively. Statistical analysis did not reveal intergroupdifferences in the severity of ischemia or the efficacy ofreperfusion.

Characterization of brain injury

Cerebral protein synthesis

Sixty minutes of intraluminal thread occlusion resulted ina suppression of global cerebral protein synthesis through-out the vascular territory of the MCA. During the recircu-lation, protein synthesis partly recovered in the cortex(12-h reperfusion), but not in the basal ganglia or in thethalamus (Figs. 2, 3).

TUNEL

After MCA occlusion, DNA fragmentations, as visualizedby TUNEL, were clearly confined to neurons and appearedlater than the inhibition of CPS (Figs. 2, 3). The numberof TUNEL-positive neurons was counted at the level ofcaudate-putamen and hippocampus (data not shown), andincidence maps were calculated to illustrate the topicaldistribution of TUNEL positivity.

At the end of 1-h ischemia, neurons were TUNEL neg-ative, but after 3 h of recirculation numerous TUNEL-pos-itive neurons were detected both in caudate-putamen andhippocampus. TUNEL positivity was restricted to the cen-tral parts of the ischemic territory, where it clearly colo-calized with the area of permanently suppressed proteinsynthesis. After 12 h, the number of TUNEL-positive neu-rons was lower, but the expansion of labeling was similar.These findings are in line with our earlier investigation ofthe evolution of ischemic cell death [14] and confirm thatrecirculation after 1 h of MCA occlusion prevents irre-versible damage in the penumbra, but not in the core ofthe primary ischemic lesion.

Regional pattern of protein changes

Histochemical evaluation of selected proteins revealed sixdifferent patterns of regulation: early or late unilateral

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Fig. 1 Measurement of blood flow in cerebral cortex of mice sub-mitted to 60-min MCAO without (A) or with 3-h (B) and 12-h (C)recirculation. Blood flow was recorded by laser Doppler flowme-try in the territory of the occluded MCA and expressed as percent-age of the pre-ischemic value (means ± SD; n=4 per group). Mea-surements were carried out during vascular occlusion and duringthe initial 15 min of reperfusion. Note abrupt decline of flow aftervascular occlusion in A–C, and return of flow to control levelswithin 15 min of recanalization in B and C (MCA middle cerebralartery, MCAO MCA occlusion)

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Fig. 2 Representative experiments showing inhibition of global CPS,TUNEL and various up- and down-regulated proteins at the end of60-min MCAO and at 3 h and 12 h of post-ischemic recirculation, re-

spectively. The topographical distribution of changes is summarizedin the incidence maps of Figs. 3, 4, 5 (CPS cerebral protein synthesis,TUNEL terminal transferase biotinylated-dUTP nick end labeling)

Fig. 3 Incidence maps of suppressed CPS and neurons positive forTUNEL on coronal sections of the mouse brains at various times af-ter 60-min MCAO. Areas of disturbed metabolism were outlined at

the level of caudate putamen (left) and dorsal hippocampus (right)and superposed to calculate the incidence of alterations as the per-centage of the number of animals (n=4 per group)

down-regulation, mainly in the basal ganglia, early or lateunilateral up-regulation both in the cortex and basal gan-glia, and bilateral cortical up-regulation with or withoutunilateral up-regulation in basal ganglia (Figs. 4, 5). Thefunctional characteristics of the regulated proteins aregiven in the Table 1.

The specificity of the immunohistochemical staining wasconfirmed in each experiment, using negative controlswithout application of the primary antibody, and in se-lected experiments by preabsorption of the primary anti-body with the immunogene blocking peptide. Diseasespecificity was confirmed by comparison with sham-oper-ated controls. Focal brain changes were evaluated bycomparing the ipsilateral hemisphere with the non-isch-emic contralateral side.

The down-regulated proteins (Nurr1, SRPK1, hPrp16,dynamin, cyclin B1 and Rag2) were constitutively ex-pressed throughout the brain except in the inhibited area ofcaudate-putamen. The dynamics of the regulation showeddifferent time patterns (Fig. 4). Sixty minutes of perma-nent focal ischemia suppressed the expression of the tran-scription factor Nurr1 mainly in the area of lateral basal

ganglia, but not that of the other proteins investigated.Following 3 h of recirculation, the expressions of fourproteins (cyclin B1, dynamin, hPrp16 and Rag2) wereinhibited and remained down-regulated at 12 h in the in-farct core. Extent and incidence of down-regulation de-clined at 12 h of reperfusion in the case of Rag2 andNurr1 in accordance with the partial recovery of CPS(Fig. 2).

SRPK1 was the only one of the 15 tested proteins toshow a histochemically detectable decline only at the longerrecirculation time. However, an intracellular translocationof the protein from the nucleus to the cytosol could al-ready be observed after 3 h of recirculation (Fig. 6). Thefinal loss of the protein in the ipsilateral side was accom-panied by a stimulation of the expression in the contralat-eral, non-ischemic hemisphere (data not shown).

The up-regulated proteins showed infarct-dependent(MAPK p49, RhoB) or -independent (CAS, FKBP12,Nek2, paxillin, p-Stat6) distribution, or a combination ofthe two (TCP-1 and Ran BP-1). The MAPK p49 signaloccurred after permanent occlusion and was localized tomotor, sensory, insular and piriform cortex of the in-farcted side. During recirculation, the area of MAPK p49expression first decreased and later extended to the lateralcaudate-putamen. RhoB immunostaining showed infarctspecificity and appeared both in the cortex and the basalganglia of the ischemic hemisphere during the recircula-tion.

Interestingly, Ran BP-1 and TCP-1 showed a constitu-tive expression profile bilaterally in the cortex, but after 3 h

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Fig. 4 Incidence maps of the topical representation of down-regu-lated proteins after 60-min MCA occlusion and various recircula-tion times (0, 3 and 12 h). The outlines of immunohistochemicalstainings are projected on representative brain sections at the levelof caudate putamen and expressed as percent group incidence foreach protein and time point (n=4 per group). Note different dy-namics of down-regulated proteins

of recirculation an ischemia-induced component of up-regulation was also observed in the piriform cortex andlateral caudate-putamen of the ipsilateral side.

Cellular association of regulated proteins

Cellular association was based on light microscopical ap-pearance of histochemically positive cells. The majorityof the proteins tested localized clearly in neurons. Only afew proteins showed immunoreactivity in cells with glialcharacteristics (Nek2, RanBP-1 in the cortex, FKBP12)but these associations require confirmation by cell-spe-cific double labeling.

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Table 1 Classification of selected proteins according to the distribution pattern after 1 h middle cerebral artery occlusion in mice

Regulation pattern Protein Function

Early unilateral down-regulation mainly in basal ganglia Nurr1 Transcription factor

Late unilateral down-regulation mainly in basal ganglia Cyclin B1 Cell cycle control, differentiationDynamin GTPaseHPrp16 Splicing factorRag2 Inflammatory protein, cell cycle controlSRPK1 Splicing factor

Early unilateral up-regulation both in the cortex and basal ganglia MAPK p49 Protein kinase

Late unilateral up-regulation both in the cortex and basal ganglia RhoB GTPase protein

Bilateral cortical up-regulation CAS GTPase protein, apoptosis, cytoskeletonFKBP12 Inflammatory protein, immunophillinNek2 Apoptosis, cell cycle control, differentiationPaxillin Cell adhesion proteinp-Stat 6 Transcription factor

Bilateral cortical up-regulation with unilateral up-regulation in basal ganglia Ran BP-1 GTPaseTCP-1 Protein folding, neurodegeneration

Fig. 5 Incidence maps of the topical representation of up-regu-lated proteins after 60-min MCAO and various recirculation times(0, 3 and 12 h). The outlines of immunohistochemical stainings areprojected on representative brain sections at the level of caudateputamen and expressed as percent incidence for each protein andtime point (n=4 per group). Note different patterns of uni- or bilat-erally regulated proteins

Subcellular localization and translocation of regulated proteins

The subcellular localization was clearly cytosolic in thecase of MAPK p49, RhoB, RanBP-1, FKBP12, Nek 2 andpaxillin. Other proteins, like hPrp16, CAS, cyclin B1 andRag2 were present in the nucleus. After ischemia SRPK1,Nurr1 and TCP-1 translocated from the nucleus to the cy-toplasm (Fig. 6). Occasionally, such translocations weredifficult to detect because the affected cells could undergopyknosis (see Fig. 6 where the size of the TCP-1-positivepyknotic cells in the ischemic hemisphere is similar tothat of the nuclei of the healthy non-ischemic side). Insuch cases, translocation preceded cell death in the core ofthe ischemic caudate-putamen.

Discussion

The present investigation stresses the complexity of pro-tein changes after focal cerebral ischemia and describesseveral new proteins that previously have not been associ-ated with the evolution of ischemic injury. Our model of1-h transient middle cerebral artery occlusion representsan experimental paradigm that is particularly sensitive tomolecular injury pathways. In fact, by selecting only ani-

mals in which recirculation was not impaired after theischemic episode, tissue damage was mainly mediated byprotracted, presumably gene expression-dependent molec-ular abnormalities.

This has been demonstrated by bioluminescence imag-ing of ATP, which upon recirculation revealed rapid re-covery of energy metabolism, followed after a few hoursby secondary energy failure [15]. The region of secondarybreakdown of energy metabolism precisely matches thatof ongoing inhibition of protein synthesis, indicating a closeassociation with delayed cell death. Interestingly, primaryrecovery of protein synthesis, which is much slower thanthe initial recovery of energy metabolism, progresses fromthe peripheral to the central parts of the recirculated vas-cular territory, whereas secondary ATP failure progressesfrom the core to the periphery [15]. Depending on thespeed of primary recovery of protein synthesis, on the onehand, and on the delay of secondary energy failure, on theother, the volume of delayed tissue injury may vary greatly[33]. As these variables depend both on the duration ofischemia and on the efficacy of post-ischemic recircula-tion, the dynamics of delayed tissue injury must be stud-ied under strictly controlled experimental conditions. Inthe present study this was achieved not only by using astandardized MCA occlusion protocol, but also by select-ing animals with the same recirculation profile, i.e., aninitial blood reperfusion rate equal to or above 100% ofcontrol.

The different recovery profiles of energy metabolismand protein synthesis lead to the dissociation of post-isch-emic gene regulation at the transcriptional and transla-tional level. During ischemia, DNA transcription and RNAtranslation are equally suppressed because both pathwaysare energy dependent. With the recovery of energy metab-olism, transcription is resumed, but the ongoing inhibitionof protein synthesis prevents RNA translation [31]. Inbrain regions with secondary energy failure, transcriptionis again suppressed, whereas in those parts where proteinsynthesis is resumed both transcription and translation re-cover. This and the possibility of superinduction of DNAtranscription in regions of disturbed translation [2, 8] cre-ate a complex pathobiochemical situation, the outcome ofwhich depends on the individual recovery profile and, there-fore, may vary under different experimental conditions.

The proteins studied in the present investigation werechosen from a list of selectively regulated genes that wepreviously established by multi-Western blot analysis ofhemispheric protein extracts [42]. The GTPases RhoB,CAS, Ran BP-1 and dynamin are molecular switches thatcontrol a wide range of signal transduction pathways. Theyare mainly known for their role in regulating the actin cy-toskeleton, but their ability to influence cell polarity, mi-crotubule organization, membrane transport pathways andtranscription factor activity is probably just as significant[7, 39].

The GTPase CAS was up-regulated bilaterally in thecerebral cortex, representing an expression pattern thatapparently is unrelated to the evolution of focal brain isch-emia. This differed from the other investigated GTPases,

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Fig. 6 Translocation of proteins SRPK1 and TCP-1 from the nu-cleus into the cytoplasm after 60-min MCAO, followed by 3-h re-circulation. Immunohistochemical stainings of striate nucleus ofthe ischemic hemisphere (A, C), and of the contralateral intacthemisphere (B, D). Note reduction of cell size in the ischemichemisphere due to beginning pyknosis

which all exhibited focal abnormalities. The associationof RhoB with impeding cell death has been described be-fore [41] and is confirmed by the present study. Here wehave demonstrated the inverse regulation of dynaminwhich is apparently down-regulated during infarct evolu-tion. Dynamins are microtubule-associated proteins withputative function in endocytosis, actin cytoskeleton, synap-tic transmission and neurogenesis [35].

Another new observation is the dual regulation of RanBP-1, which exhibits both infarct-dependent and infarct-independent expression after focal ischemia. Beside a bi-lateral constitutive expression in the cortex, a Ran BP-1signal appeared temporarily in the ipsilateral basal gangliaafter 3 h of recirculation. This protein is localized in thecytoplasm and enhances the Ran GTPase activating pro-tein (RanGAP) activity, which plays a role in the nucleo-cytoplasmic transport. Ran is implicated in diverse cellu-lar processes including DNA replication, entry into andexit from mitosis and the transport of RNA and proteinsthrough the nuclear pore complex. Active RanGTP andinactive RanGDP are tightly regulated by guanine nu-cleotide exchange factors (GEFs) and GTPase activatingproteins. Ran BP-1 binds specifically to the GTP-boundform of Ran and acts as an inhibitor of RanGEF function[4, 39]. Its up-regulation may reflect a temporary induc-tion of ischemia-related transport mechanisms.

The regulation of these four GTPase proteins clearlyfollows different dynamics and shows different spatialdistributions after the ischemia/reperfusion injury. Somestudies have established a role of Rho GTPases in con-trolling the enzymatic activity of serine-threonine kinasesknown as mitogen-activated protein kinases (MAPKs) [41].The new member of this enzyme family, MAPK p49, wastested in our study. It presented an early up-regulation inthe preserved peri-infarct zone during and following isch-emia, presumably taking part in the neuroprotection of thesurviving neurons of the penumbra.

Another class of functional proteins examined in thisstudy are the transcription factors Nurr1 and p-Stat6. Theorphan nuclear receptor Nurr1 is encoded by an interme-diate early gene that can be rapidly induced by differentstress conditions. Nurr1 shows a distribution similar to thatof dopamine-containing neurons [37] and is a potentialregulator of gene expression of dopaminergic phenotypicmarkers. Gene coding for dopamine transporter and thedopamine biosynthetic enzyme tyrosine hydroxylase havebeen demonstrated to respond to Nurr1 [37]. The induc-tion of Nurr1 mRNA following permanent focal ischemia[20] and global cerebral ischemia [19] has also been de-scribed. In our experiments the early down-regulation inthe infarcted hemisphere is either due to post-translationalprotein modification or indirectly to the lack of energysupply. The translocation of Nurr1 in the hypoxia-dam-aged striatal cells from the nucleus to the cytosol is anovel finding that has not been reported before.

P-Stat6 was expressed in the cortex of both hemi-spheres with low intensity. The phosphorylation of Stat6did not show an infarct-dependent profile in our experi-ments. Stat6 mediates signals for IL-4 and possibly IL-13,

and is thought to modulate the basal transcription machin-ery by binding to p300/CBP [21].

To the best of our knowledge, the ischemia-inducedregulation of the cell cycle control proteins Rag2 and Nek2has not been described before. However, the role of cyclinB1, another cell cycle regulator, in neuronal cell death fol-lowing retinal ischemia/reperfusion [26] or transient MCAocclusion [17] has already been investigated. It was con-cluded that aberrant expression of cyclin B1 may play arole in necrotic cell death after retinal ischemia, but not inapoptosis associated with stroke. In our study, cyclin B1and Rag2 were down-regulated in the injured basal gan-glia during the reperfusion. In contrast, Nek2 was up-reg-ulated bilaterally in the cortex during and after the MCAocclusion.

Cyclin B1 is mainly expressed in the G2/M phase ofcell division which, in turn, is triggered by the cyclin B1-Cdc2 complex. In the M phase; cyclin B1 is phosphory-lated in the cytoplasmic retention sequence, which is re-quired for nuclear export. During the interphase, cyclinB1 shuttles between the nucleus and the cytoplasm be-cause constitutive nuclear import is counteracted by rapidnuclear export [12]. Entry into mitosis requires not onlythe presence of cyclin-dependent proteins, but also a sec-ond, structurally distinct serine/threonine kinase known asNIMA. NIMA-related kinases like Nek2 investigated hereare emerging as critical regulators of centrosome structureand function [10]. Similarly, the recombination activatorgene Rag2, which is essential for immunoglobulin pro-duction [1], may provide new information on cell cycle-associated mechanisms of ischemic injury.

A novel observation is also the ischemia-dependent reg-ulation of the splicing factors SRPK1 and hPrp16. Reversiblephosphorylation plays an important role in pre-mRNAsplicing in mammalian cells. SR-protein specific kinase(SRPK1) has been shown to phosphorylate the SR familyof splicing factors [46]. According to our results, the inhi-bition of oxygen supply caused translocation of SRPK1from the nucleus to the cytosol, blocking its nuclear func-tion, and later led to its complete down-regulation in thefocus of the infarcted area. HPrp16 is involved in the sec-ond step of splicing when the exons are fused with con-comitant release of the intron lariat [49]. The post-isch-emic down-regulation described here suggests disturbancein splicing activity, which is in line with our previous ob-servations on the splicing regulating protein tra2-beta1 [6].

The behavior of the immunophilin FKBP12 (FK506binding protein12) following focal cerebral ischemia hasbeen investigated by Kato et al. [22]. They found thatFKBP12 immunoreaction decreased rapidly in the isch-emic core and increased in the penumbra after 4-h recir-culation. We observed a peripheral up-regulation in bothischemic and healthy cortex. FKBP12 plays an importantrole for fate decisions between neuronal survival anddeath following cerebral ischemia and is involved in in-flammatory reactions.

The post-ischemic up-regulation of TCP-1 (t-complexpolypeptide) in the ipsilateral basal ganglia, is another in-teresting finding that may be linked to the recently dis-

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covered role of endoplasmic reticulum (ER) dysfunctionin the evolution of ischemic injury [34]. TCP-1 is a chap-erone for the folding of tubulin [48], whose assembly intomicrotubules is critical to many cellular processes. As ERdysfunction results in disturbances of protein folding, up-regulation of TCP-1 may be a rescue response in analogyto the similarly up-regulated chaperone protein HSP70[14].

Finally, the cell adhesion protein paxillin showed mul-tifocal up-regulation in cerebral cortex, which may be ofimportance for controlling the recently documented long-distance migration of endogenous [3] or transplanted stemcells [18] to the ischemic lesion.

In summary, the combination of metabolic and molec-ular markers of ischemic injury with the immunohisto-chemical evaluation of biologically important regulatoryproteins provides precise information on the associationand disassociation between selective gene expression anddisease evolution. Our observations stress the importanceof regional analysis for the interpretation of genomic andproteomic data, and they contribute to the identification ofnew regulatory pathways that may be involved in the evo-lution of post-ischemic brain injury. Obviously, furtheranalysis of these pathways will be necessary to specifytheir importance as new targets for neuroprotective drugdesign.

Acknowledgements This study was supported by grants from theEuropean Community (Framework V, StrokeGene Consortium,contract number QLG3-CT-2000–00934) and from the GermanMinistry of Education and Research (Kompetenznetwerk Schlag-anfall, Projekt B1). The authors are grateful to A. Janz, M. Jagod-nik and P. Janus for excellent technical assistance, B. Huth for theartwork and A. Lorig for copy editing of the manuscript.

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