Journal of the Neurological Sciences 320 (2012) 110–117

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Evolution of microglial activation in ischaemic core and peri-infarct regions afterstroke: A PET study with the TSPO molecular imaging biomarker [

11C]vinpocetine

Balázs Gulyás a,⁎, Miklós Tóth a, Martin Schain a, Anu Airaksinen a,1, Ádám Vas b, Konstantinos Kostulas c,Per Lindström d, Jan Hillert c, Christer Halldin a

a Karolinska Institutet, Department of Clinical Neuroscience, Psychiatry Section, S-171 76 Stockholm, Swedenb Gedeon Richter Plc., H-1103 Budapest, Hungaryc Karolinska Institutet, Department of Neurology, Neuro-angiological Research Center, Karolinska University Hospital Huddinge, S-141 86 Huddinge, Swedend Karolinska Institutet, Neurology Section, Department of Clinical Neuroscience, S-171 76 Stockholm, Sweden

⁎ Corresponding author at: Karolinska Institutet, Depscience, Psychiatry Section, Karolinska University HosStockholm, Sweden. Tel.: +46 8 51770910; fax: +46 8

E-mail address: balazs.gulyas@ki.se (B. Gulyás).1 Present address: Department of Chemistry, Univ

A221, Helsinki, Finland.

0022-510X/$ – see front matter © 2012 Elsevier B.V. Aldoi:10.1016/j.jns.2012.06.026

a b s t r a c t

a r t i c l e i n f o

Article history:Received 24 January 2012Received in revised form 3 May 2012Accepted 23 June 2012Available online 27 July 2012

Keywords:Positron emission tomography (PET)Radioligand[11C]vipocetine

Peripheral benzodiazepine receptor (PBR)18 kD translocator protein (TSPO)StrokeIschaemic corePeri-infarct zoneMicrogliaMolecular imaging biomarker

Although there is increasing evidence for microglial activation after an ischaemic stroke in the infarct coreand the peri-infarct region, the “evolution” of the process in stroke patients is poorly known. Using PETand [

11C]vinpocetine, we measured the regional changes of TSPO in the brain of nine ischaemic stroke patients

up to 14 weeks after the insult. Already a week after stroke there was an increased radioligand uptake, indi-cating the up-regulation of TSPO and the presence of activated microglia, in both the ischaemic core and theperi-infarct zone. This increased activation showed a steady decrease with post stroke time. The proportionbetween %SUV values in the peri-infarct zone and the ischaemic core increased with time. There were notime-dependent TSPO activity changes in other regions, not affected directly by the stroke. The present obser-vations demonstrate that increased regional microglia activation, as a consequence of stroke, can bevisualised with PET, using the TSPO molecular imaging biomarker [

11C]vinpocetine. The evolution of this

microglial activation shows a time dependent decrease the gradient of which is different between theperi-infarct zone and the ischaemic core. The findings indicate an increased microglial activation in theperi-stroke region for several weeks after the insult.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

The search for specific molecular imaging biomarkers related toneuroinflammation and neurodegeneration has in recent years beena major endeavour in PET radiotracer research. Neurological diseases,entailing neuronal death, neurodegeneration and neuroinflammation,offer various target systems for imaging biomarkers. One of the mostcommonly considered such systems is the 18 kDa translocatorprotein (TSPO), or, as earlier called, the peripheral benzodiazepine re-ceptor (PBR) system. TSPO is up-regulated in the human brain duringnormal ageing as well as in diseases entailing regional or globalneuroinflammatory processes in the brain, such as multiple sclerosis,stroke, acute brain trauma, Alzheimer's disease or Parkinson's disease[1–3].

Under normal circumstances, the TSPO density is not particularly highin the human brain. During normal ageing as well as during pathological

artment of Clinical Neuro-pital Hus R5:02, S-171 7651771753.

ersity of Helsinki, PL 55,

l rights reserved.

processes, including neuroinflammation, neurodegeneration and acutebrain disorders, the expression of TSPO increases, primarily due to the ac-tivation ofmicroglia [1–3]. The activation ofmicroglia and the consequentup-regulation of the TSPO system have been demonstrated in the “nor-mal” ageing brain [4–8] as well as in various CNS diseases with concomi-tant neuroinflammation, including Alzheimer's disease, Parkinson'sdisease, ALS, multiple sclerosis, stroke and traumatic brain injury [9,10].

During the past years numerous molecular imaging biomarkersfor the TSPO have been tested using positron emission tomography(PET) [11]. Among available PET radioligands for diagnostic orpre-clinical PET imaging, [

11C]PK11195 is considered the “golden

standard” [12–17]. Several novel TSPO ligand candidates, due to var-ious factors including low blood–brain-barrier penetration, insuffi-ciently low in vivo binding potential, binder–non-binder differences,etc., failed to yield acceptable outcome measures in order to usethem as diagnostic markers, whereas others are in various phases ofpre-clinical and clinical testing, including [

11C]PBR28, [

11C]DAA1106,

[18F]DAA1106, [

18F]DAA1106, [

11C]DPA713 and [

18F]DPA714 [18–25].

Earlier PET imaging studies in cerebral hypoxic lesions and strokehave mainly utilised the “working horse” of clinical diagnostic PETimaging, FDG, as tracer [26–29]. More recently, realising the presenceof neuroinflammation in stroke, molecular imaging biomarkers of the

Table 1The patients' basic data. As all patients were male, the gender is not indicated in thetable. The post stroke time indicates the period between the last stroke and the PET in-vestigation in days.

No. Age(year)

Injectedradioactivity(MBq)

Post stroketime (days)

Stroke region(s)

1 72 256 7 Right caput nucleus caudatus2 70 390 10 Left thalamus3 19 133 28 Right primary visual cortex4 61 361 31 (1) Left putamen-corona radiata;

(2) Left frontal lobe,periventricularly

5 69 377 35 (1) Right temporal and frontallobes next to sulcus Sylvii;(2) Right hemisphere white matter,posterior to temporal horn

6 72 389 40 Right gyrus precentralis7 61 337 55 Left cella media8 66 121 75 Left temporal lobe9 35 272 98 Left hemisphere, rostral to the

lateral ventricle

111B. Gulyás et al. / Journal of the Neurological Sciences 320 (2012) 110–117

TSPO system have come to the fore, including PK11195 [16,28,30–34],DPA714 [24], FEAC and FEDAC [35]. Using [

11C]PK11195 a number of

earlier studies aimed at exploring the presence of activated microgliain and around the infarct region as well as exploring the temporal dy-namics of microglial activation. The studies indicated that within afew days after the insult activated microglia appears in theperi-infarct zone and a few days later in the ischaemic zone, as well.The activation reaches a peak value approximately 1 week after thestroke and thereon it decreases [17]. Activation in the peri-infarctzone appears to be more intense than in the ischaemic core[16,17,34]. Despite these studies, the detailed temporal dynamics ofthe stroke related neuroinflammation, with special regard tomicroglial activation, is not known in detail.

Vinpocetine (ethyl apovincaminate) is a synthetic compound re-lated to the Vinca minor alkaloid, vincamine [36]. Early autoradio-graphic studies on whole hemisphere human brain slices with [

11C]

labelled vinpocetine have indicated that the radiolabelled compoundshows a moderate binding to cortical and sub-cortical brain struc-tures and this binding can be suppressed by the excess of the coldcompound. The study indicated that vinpocetine may bind to recep-tors and/or transporters in the human brain with moderate affinity[37]. Later on, both its receptor affinity characterization (IC50 forTSPO: 200 nM) and a number of investigation with PET havesupported the fact that radiolabelled vinpocetine may serve as aTSPO biomarker [38]. Although it binds with a moderate affinity toTSPO, compared to ligands with nanomolar–sub-nanomolar bindingaffinities to TSPO, due to its favourable brain penetration it can be ausable PET radioligand for in vivo exploration of the TSPO system inhuman brain [39].

Using positron emission tomography (PET) we have demonstrat-ed in both non-human primates and humans that [

11C]vinpocetine

may act as a prospective radioligand for TSPO, and, consequently, itmay be used as a biological marker in PET studies to label in vivothe TSPO system [38,40–42]. Comparative analysis of novel TSPOPET radioligands also indicates that, despite its relatively lower bind-ing activity to TSPO, [

11C]vinpocetine may have comparative advan-

tages [11,43]. Compared to [11C]PK11195, the advantages of [

11C]

vinpocetine appear, among others, in higher brain uptake and higherregional binding potential values in various brain structures, withspecial regard to those regions involved in stroke [44]. The hypothesisthat [

11C]vinpocetine can serve as a molecular imaging biomarker of

TSPO has successfully been tested and demonstrated by us in earlierPET studies in sclerosis multiplex patients [45] as well as in stroke pa-tients [8,44].

The main objective of the present clinical PET study was to explorethe temporal dynamics of microglial activation in stroke patientsusing [

11C]vinpocetine as a molecular imaging biomarker of TSPO. In

nine stroke patients the global and regional uptake values, includingthose in the ischaemic core region and the peri-infarct zone andexpressed as %SUV, of [

11C]vinpocetine were measured and their cor-

relation with post stroke time was analysed.

2. Methods

2.1. Subjects and ethical committee approval

Twelve male stroke patients, capable to walk, to visit the PET cen-tre and to participate in the study without medical help, wererecruited from either the Neurology Department, Karolinska Univer-sity Hospital, Solna or the Neurology Department, Karolinska Univer-sity Hospital, Huddinge, Sweden.

The patients were informed both verbally and in written formabout the objectives, procedures and eventual risks of the experi-mental procedure, in line with the Helsinki Declaration, and theyhave signed an informed consent. The investigation was conductedin line with the Karolinska Institute's regulations regarding clinical

experiments, and permissions were obtained from the appropriateEthical and Radiation Safety committees of the Karolinska Institute(Dnr. 03–385).

Two patients started the PET examination but stopped the PETscan in early phase due to phobic feelings in the scanner. Their pre-liminary patient data are not included here. The experiments weresuccessfully performed on ten patients. In one patient the MRI scanindicated an intracranial tumour which was confirmed by consecu-tive investigations following the PET scan; for this reason the patient'sdata were not used for further analysis. I.e. the final analysis com-prised data obtained in nine stroke patients. The age of the patientswas between 19 and 72 years (average age: 58±19 years). One ofthem was scanned in the acute phase (1–7 days), one in thesub-acute phase (7–21 days), and seven in the chronic phase (be-tween 21 and 98 days). A short summary of the patients is shownin Table 1.

2.2. MRI scans

In each case MR images (T1, T2, PD) were made on the patients ona GE Signa 1.5 T scanner. The images were analysed by a neuroradiol-ogist as well as an independent expert observer. The location and ex-tent of the stroke region(s) were identified in the T1 images;sometime with the help of the T2 or PD images.

2.3. Radiochemistry

All the chemicals used for radiosynthesis were purchased fromcommercial suppliers and used without further purification.Vinpocetine and the starting material apovincaminic acid were pro-vided by Gedeon Richter Plc., Budapest, Hungary. The synthesis wasperformed according to the method previously published [42]. Thespecific radioactivity obtained at the time of injection of [

11C]

vinpocetine was higher than 2.8 GBq/μmol, corresponding to a totalmass injected of less than 20 μg. The radiochemical purity was betterthan 99%.

2.4. PET measurements

The PET measurements were made in 3D acquisition mode on aSiemens ECAT EXACT HR scanner, with 47 image planes placed at3.125 mm from each other. The scanner's in-plane resolution(FWHM) is 3.8 mm. The acquisition time was 66 min, consisting of15 frames (3×1 min, 3×3 min, 9×6 min). Attenuation correctionwas calculated, using 10 min transmission scans in each subject

Fig. 1. Examples of the determination of disease specific ROI's. Patient No. 4 (see Table 1). A: PD (proton density) MRI scan, B and C: T1 weighted MRI scan. D: [11C]vinpocetine PET

scan. Summation image (acquisition time between 9 and 66 min after radioligand injection). Horizontal slices, x=+15 mm (HBA coordinates). Arrows in A and B indicate the le-sion site, the contours of the stroke region in C and D are in red, the peri-stroke region in black. Post mortem time: 31 days.

112 B. Gulyás et al. / Journal of the Neurological Sciences 320 (2012) 110–117

with a68Ge source. Four subjects were equipped with individually

moulded head fixation helmets during the PET scans [46]. In six sub-jects the head fixation was provided with a plastic head holder andelastic strips limiting head movements. The average i.v. injected ra-dioactivity dose with [

11C]vinpocetine was 288±101 MBq (range:

121–390 MBq) (see Table 1).Following image reconstruction, the MRI and PET images were

further processed and analysed in the Karolinska Institute's HumanBrain Atlas (HBA) system [47]. Further the co-registration of the indi-vidual MRI and PET images, for analysis of global regional radioactiv-ity in the human brain, standard volumes of interest (ROI's) weredrawn on the reconstructed PET images for all major anatomical re-gions of the brain, using at least two or more image sections foreach ROI. The anatomical delineation of ROI's was guided by theHBA, containing the templates of over 200 neuroanatomical struc-tures. In the present paper x, y or z values were given in HBA coordi-nates, which, practically, coincide with the Talairach coordinates [48]the only difference being the reference brains' original sizes.

Using the HBA, ROI's were drawn on the reconstructed PET-imagesfor the following “standard” brain regions: whole brain, cerebellum,centrum semiovale (one–one regions), thalamus, striatum, frontal, pari-etal, temporal and occipital lobes (left and right regions, respectively).Using the MRI scans (T1, T2, PD) as basis, and the corresponding PETscans, and complimentary information, individual “disease-specific”ROI's (ischaemic core and peri-infarct zone) were also created by twoindependent observers (BG and MT), followed by a consensus meetingto identify the chosen ROI's which covered, in the first instance, the

0

0.5

1

1.5

2

2.5

3

0 10 20 30 40 50 60 70

% o

f to

tal i

nje

cted

rad

ioac

tivi

ty

Time (min)

Fig. 2. Average brain uptake curves of [11C]vinpocetine, expressed in % of total injected

radioactivity. Error bars indicate SD values.

ischaemic core region and, consequently, the peri-infarct zone (Fig. 1).Whereas the delineation of the core regions could effectively be donewith the help of the MRI scans, for the delineation of the peri-infarctzones the PET images were often required in addition to the MRIscans. In the PET images increased regional contrasts (in either direc-tion) in uptake values in the immediate neighbourhood of the infarctcore guided the two observers in the delineation of the peri-infarctzone. In two patients two ischaemic core regions and relatedperi-infarct zones were identified in an identical manner as describedabove and the corresponding uptake valueswere used for further calcu-lations. In these cases in the figures and tables, the averages of the tworegional values are indicated.

Regional radioactivity was determined for each frame and plottedversus time. In line with our laboratories' experience [49,50], the sum-mation images were based on the sampling period between 9 min and66 min after tracer administration, in order to avoid the contribution ofthe initial “flow phase” of the brain uptake [38,42,44,45].

Total radioactivity values (nCi/ml) in the whole brain, the afore-mentioned “standard” brain regions, and the “disease-specific re-gions” (ischaemic core and peri-infarct zone) were obtained bycalculating the average radioactivity concentration in a ROI. This hasgiven rise to the measurements of global brain uptake values of theradioligand, expressed as % of the total injected radioactivity. As anext step, in the ROI's percentage standardised uptake values(%SUV) were calculated. The %SUV was defined by the following for-mula, wherein radioactivity concentrations were decay corrected andnormalised to injected radioactivity and body weight:

%SUV ¼ the radioactivity in the target Bq=gð Þtotal injected radioactivity ðBqÞ=bodyweight gð Þ � 100 ð1Þ

In line with a recent detailed analysis of [11C]vinpocetine PET data

obtained in healthy volunteers and Alzheimer's disease subjects [8],the %SUV values were used as main outcome measures of the presentinvestigation.

We considered the choice of %SUV as the ‘main outcome measure’due to the fact that the choice and definition of a satisfactory refer-ence region for TSPO radioligands has been a challenging issue inrecent literature (see e.g. [51]) and despite its relative drawbacks(vulnerability to noise, etc.), it appears to be a robust and solid quan-titative measure. In our previous studies we used the cerebellum asan “operationally defined reference region”, but not as a preferredand established reference region for vinpocetine and other TSPOradioligands [38,45].

To complement the %SUV value as the ‘main outcome measure’, wehave also calculated and analysed binding potential (BPND) values in themain cortical and sub-cortical regions as well as in the ischaemic core

Table2

Region

al%S

UVva

lues

(ave

rage

±SD

)in

differen

tbrainregion

s.(A

ve:av

erag

e,SD

:stan

dard

deviation;

I:ipsilateralh

emisph

ere;

C:co

ntralateralh

emisph

ere.)

Region

Fron

tal

Tempo

ral

Parietal

Occipital

Caud

ate

nucleu

sPu

tamen

Thalam

usCe

rebe

llum

Stroke

region

sin

theaffected

side

Intact

coun

terpartregion

sin

the

contralaterals

ide

Side

IC

IC

IC

IC

IC

IC

IC

Isch

aemic

core

Peri-infarct

zone

Isch

aemic

core

Peri-infarct

zone

Ave

99.25

99.97

103.36

102.26

98.01

99.20

114.87

114.82

94.77

107.23

115.45

122.41

114.32

118.04

98.99

90.51

118.42

99.89

108.10

SD12

.81

14.01

17.17

17.71

16.89

15.77

18.68

19.72

36.63

20.23

29.33

28.90

19.53

24.01

22.53

32.75

32.04

27.07

24.86

113B. Gulyás et al. / Journal of the Neurological Sciences 320 (2012) 110–117

and the peri-infarct zone in both the affected side and their intact coun-terparts in the contralateral side. The calculation of the BPND values wasdescribed in detail in Cselényi et al. [52,53]. They were estimated byusing the reference region version of Logan's graphical analysis [54]with the ipsilateral cerebellum as reference region, as suggested by var-ious authors [55,56], to avoid the effect of cerebellar diaschisis [57]. Sta-tistical comparisons between group data were performed withStudent's t-test (paired difference or independent two samples withequal size and variance, depending upon the given comparisons).Also, the time dependence of the %SUV in disease specific regions wasanalysed. However, in order to reduce the effect of individual variationsin brain uptakes of the radioligand, the quotient between %SUV values,obtained in the affected regions, to the cerebellar %SUV values wereused, and analysed using linear regression analysis.

3. Results

3.1. Brain volumes

In order to explore whether possible volumetric changes in thebrain, as a result of the stroke, may influence the measurements, wecompared the brain volumes of the stroke patients with that of agematched healthy volunteers, as well as the volumes of the affectedand non-affected hemispheres with each other in the stroke patients.Despite the preceding stroke(s) there was no significant difference in“netto” brain volumes (grey matter and white matter in cerebral andcerebellum, without the third and forth ventricles) between the pa-tient group and an age matched control group of nine healthy volun-teers (age range: 25–78 years; average age: 57±19 (SD) years),taken from our laboratories' “control subject database” (average vol-ume: 1293±196 (SD) ml and 1221±116 (SD) ml, stroke patientsand age matched controls, respectively. In the stroke group therewas no significant difference between the volumes of the affectedhemispheres and the non-affected hemispheres (593±108 (SD) mland 585±99 (SD) ml, respectively). A pair-wise comparison of thevolumes of the hemispheres in each patient revealed that the affectedhemisphere was slightly (8±21 (SD) ml) larger that the non-affectedone, the difference being less than 1% of the total brain volume)

3.2. Global brain uptake and disposition of [11C]vinpocetine

[11C]Vinpocetine entered the brain and reached a peak uptake ac-

tivity value in the brain after 1 min. In stroke patients the peak uptakevalues of the nine patients investigated with [

11C]vinpocetine was on

the average 2.12±0.48 (SD) % of total injected radioactivity (Fig. 2),the average brain uptake values in the summation images(9–66 min) was 1.50±0.35 (SD).

3.3. Regional %SUV values

Regional %SUV values are displayed in Table 2. For “standard” regionsthe values are grouped along the affected (ipsilateral) and non-affected(contralateral) hemispheres, whereas the “disease-specific” regionsrepresent the stroke regions (ischaemic core and peri-infarct zone inthe affected hemisphere) and their intact counterparts in the contralat-eral hemisphere. In line with our earlier observations [38,41,42], thehighest up-take values were found in the putamen and thalamus,followed by the occipital cortex and other cortical regions. The averageup-take in the ischaemic core was lower than the average global brainup-take, whereas that in the peri-infarct zone was higher than the aver-age brain up-take and reached the level of the %SUV values obtained inthe thalamus and striatum. There were significantly higher %SUV valuesin the peri-infarct zone (118.42±32.05) than in the ischaemic core(90.51±32.75) in the overall patient population (pb0.05). A similar dif-ference in the intact counterpart regions in the contralateral hemispherewas not present, the corresponding values being 108.10±24.86 and

Fig. 3. Typical examples demonstrating ischaemic core and peri-infarct zoneradioligand uptake in patients with short post and long post stroke times. A: PatientNo. 1, post stroke time: 7 days. B: Patient No. 9, post stroke time: 98 days. Circles indi-cate the ischaemic core, arrows the peri-infarct zone.

Fig. 4. Average time activity curves (TAC's) of %SUV uptake in the infarct zone and theperi-stroke region in the ipsilateral (affected) side (A) and the contralateral(non-affected) side (B).

114 B. Gulyás et al. / Journal of the Neurological Sciences 320 (2012) 110–117

99.89±27.07, respectively. Typical examples demonstrating ischaemiccore and peri-infarct zone radioligand uptake in patients with shortpost and long post stroke times are displayed in Fig. 3.

3.4. Regional BPND values

Regional BPND values are shown in Table 3. In line with earlier ob-servations [38,41,42], the binding potential of [

11C]vinpocetine is very

moderate and is the highest in the thalamus, the putamen and the oc-cipital lobe, as seen in the present patient population, as well. Al-though there were no significant differences between the BPNDvalues obtained in the affected regions in the ipsilateral and the con-tralateral sides, in both the ischaemic core and the peri-infarct zone inthe affected (ipsilateral) side the average BPND values were markedlyhigher (0.32 and 0.25) than those obtained in their intact counter-parts (0.13 and 0.17) in the contralateral (non-affected) hemisphere.

3.5. Time activity curves of radioligand uptake in the affected regions

The time activity curves (TAC) of radioligand uptake in theischaemic core and the peri-infarct zone in the affected hemisphereas well as in their intact counterpart regions in the non-affectedhemisphere are shown in Fig. 4. There were significant differences be-tween the TAC's of the ischaemic core and of the peri-infarct zone inthe affected hemisphere up to the 40th minute after radioligand ad-ministration. On the other hand, there were no significant differencesbetween the TAC's belonging to the intact counterpart regions in thenon-affected hemisphere, between the TAC's of the peri-infarct zonein the affected side and that of its intact counterpart region in thenon-affected side, and between the TAC's of the ischaemic core inthe affected side and the TAC of the mirror-symmetric counterpartof the ischaemic core in the non-affected hemisphere.

3.6. Post-stroke time and %SUV values

The ratios of the individual %SUV values in the ischaemic core andthe peri-infarct zone to the cerebellar %SUV values of the nine patients

Table 3Regional BPND values (average±SD) in different brain regions. (Reference region: ipsilateralateral hemisphere.)

Region Frontal Temporal Parietal Occipital Caudatenucleus

Putamen

Side I C I C I C I C I C I C

Ave 0.10 0.13 0.15 0.13 0.13 0.15 0.23 0.15 0.13 0.21 0.25 0.28SD 0.12 0.18 0.14 0.15 0.14 0.19 0.22 0.13 0.16 0.18 0.20 0.22

are shown in Fig. 5. As the PET scans were made at various post stroketimes in nine different patients, the present graph is not representingthe temporal evolution of microglial activation in the affected regionsin the same patients, but it displays “individual snapshot of the statusof neuroinflammation” across a patient population scanned at variouspost stroke time points. The %SUV ratios were systematically higher inthe peri-infarct zone than in the ischaemic core regions, indicatingthat the activation of microglia is predominantly in the peri-infarctzone.

The slopes in Fig. 5 were not significantly different from zero for ei-ther the ischemic core or the peri‐infarct zone. However, a trend to-wards temporal decline was observed in the ischemic core (p=0.14).The effect was less pronounced in the peri-infarct zone (p=0.43). Theresiduals were normally distributed for both zones indicating that ahigher order model fitting would not provide additional accuracy.

l cerebellum; Ave: average, SD: standard deviation; I: ipsilateral hemisphere; C: contra-

Thalamus Affected regions in the ipsilateralside

Intact counterpart regions in thecontralateral side

I C Ischaemic core Peri-infarct zone Ischaemic core Peri-infarct zone

0.27 0.23 0.32 0.25 0.13 0.170.20 0.16 0.29 0.21 0.17 0.19

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 20 40 60 80 100

Fig. 5. The ‘ischaemic core:cerebellum’ and the ‘peri-infarct zone:cerebellum’ %SUV ra-tios in the nine patients (y axis), scanned at various post stroke times (expressed indays; x axis). Closed squares and solid line: linear regression of the ischaemic coredata points, y=−0.0044x+1.0971, R²=0.2813; open squares and dotted line: linearregression of the peri-infarct zone data points, y=−0.0032x+1.3542, R²=0.0905.

115B. Gulyás et al. / Journal of the Neurological Sciences 320 (2012) 110–117

4. Discussion

Neuroinflammation is a common response to numerous brainstressors of various origin, including brain trauma, stroke, hypoxia,excessive deposition of misfolded proteins or infection. A major ele-ment of neuroinflammation is the activation of resident microglialcells. Microglial activation, which includes cell proliferation, phagocy-totic transformation of the cells as well as up-regulation of the TSPOsystem in the mitochondrium, can take place regionally, in andaround localised brain lesions, or, in the case of generalised insults,globally in the brain.

The underlying mechanisms and the temporal dynamics ofmicroglial activation in in vitro stroke models as well as in animalstroke models has in recent years been studied intensively. In the in-farct region microglial activation starts hours after insult and the acti-vated microglial cells transform into phagocytes within days afterinjury [58]. Already 48 to 73 h after the injury, the resident microglialpopulation in the affected region shows an intensive proliferating ac-tivity [59]. The microglial activation is not showing identical dynam-ics in the ischaemic core and the peri-infarct zone: whereas activatedmicroglia appears within 3 days after injury in the boundary zone ofthe infarct, in the core region it is observed only from the seventhday on [60], whereas it was absent until that time [28]. In additionto the activation of the local microglia, a robust microglialinfiltration to the affected region is also a major player in localneuroinflammation in stroke which in the peri-infarct zone reachesits peak 48–72 h following insult [61]. In animal stroke models thepresence of activated microglia was demonstrated up to severalmonths after middle cerebral artery occlusion [62–64], indicating along term neuroinflammatory process after stroke.

With the advancement of molecular imaging techniques, the studyof the temporal dynamics of neuroinflammation in stroke patients,with special regard to microglial activation, has beenmade possible. Al-though various prospective TSPO radioligands have been explored inanimal stroke models [19,43,64–68], until today the “golden standard”of TSPO imaging, [

11C]PK11195 has been most widely used in human

PET studies. PET measurements using [11C]PK11195 have revealed the

presence of activated microglia in and around the stroke region anddemonstrated a difference in radioligand uptake between the ischaemiccore and the peri-infarct zone. They have also indicated a temporal dy-namics of microglial activation. In six patients Gerhard et al. [16] coulddemonstrate already 3 days after stroke onset the presence of activatedmicroglia in and around the stroke region. The radioligand binding inand around the stroke region is clearly established by the end of thefirst week, it may increase further, followed by a continuous decreaseuntil the end of the period examined (150 days in one patient). Infour ischaemic stroke patients using [

11C]PK11195 PET measurements

Price et al. [17] could find minimal radioligand binding within 72 hafter infarction in the brain. After the third day and within a weekafter the stroke the binding raised significantly in the infarction core,the peri-infarct zone and in the contralateral hemisphere. By the fourthweek some reduction in binding was observed. In a study on 18 strokepatients, studied between 2 weeks and 6 months post stroke time,Thiel et al. [34] found an initial increase, followed by a decrease, inlocal microglial activation.

Whereas our present observations support the earlier findings,obtained with [

11C]PK11195 PET in stroke patients, they complement

our existing knowledge on stroke related neuroinflammation withsignificant novel information, with special regard to the temporalchanges and the regional characteristics of microglial activation.

The temporal evolution of neuroinflammation, more specifically,the activation of microglia, in stroke patients have until today onlybeen studied at limited time points [16,17,34]. In the present investi-gation we measured [

11C]vinpocetine uptake at nine different time

points between 1 week and 14 weeks after stroke. Despite the factthat the measurements were made on various individuals and, conse-quently, the data may show variability across the patient population,they indicate a clear trend regarding the presence of microglial acti-vation in and around the stroke region early on after stroke and a de-crease of it with time. There was a significant negative correlationbetween post stroke time and the %SUV values in both the strokeand peri-stroke regions. Also, there were significant differences be-tween %SUV values in the stroke region and the combined strokeand peri-stroke region in the patient group with less than 40 dayspost stroke time and the patient group with equal or more than40 days post stroke time. All these observations, together with avail-able literature data on the evolution of microglial activation afterstroke indicate an increased initial radioligand uptake in the affectedregion, followed by a steady decrease. Consequently, the data indicatethat 1 week after the stroke there is already an increased microglialactivity in the stroke regions, including both the ischaemic zone andthe peri-infarct zone. Our present observations in human patientsare similar to those obtained in animal stroke models [38,60]. Where-as the microglial activation decreases steadily with time, there is stillactivated microglia present in the peri-infarct zone months after theinsult, in line with earlier observations [16,34].

At the same time the data indicate temporally different develop-ments of the increased microglial activities in the ischaemic coreand the peri-infarct zone, consequently, a different temporal dynam-ics of microglial activation. In the ischaemic core the slope of the de-crease in steeper than in the peri-infarct zone. This is in line withother observations, indicating the insult results in neuronal death inthe ischaemic core. On the other hand microglial activation remainsmore persistent in the peri-infarct region, indicating an ongoingmechanism aiming partly at spatially stipulating the damage zoneand partly at repairing the tissue in the immediate neighbourhoodof the infarct core.

The TAC's obtained in the ischaemic core and peri-infarct zone in theipsilateral (affected) hemisphere and the TAC's obtained in the afore-mentioned regions intact counterpart regions in the non-affected (con-tralateral) hemisphere indicate that the radioligand's uptake in theischaemic core is significantly lower in the first 40 min in this regionthan in the peri-infarct zone or in the intact counterpart regions. Onthe other hand, the %SUV values indicate that there were markedlyhigher %SUV values in the peri-infarct zone than in the ischaemic corein the affected hemisphere. Regarding the corresponding BPND values,it appears that in the affected side in the ischaemic core and theperi-infarct zone these values were moderately, but not significantly,higher than in the intact counterpart regions. Taken together thesedata, we can hypothesise that the increased %SUV values in theperi-infarct zone is partly due to an increased flow component, i.e. anincreased blood–brain barrier penetration of the radioligand, and partlydue to an increased binding to activated microglia.

116 B. Gulyás et al. / Journal of the Neurological Sciences 320 (2012) 110–117

The %SUV value changes in the ischaemic core and the peri-infarctzone during the post-stroke period covered by the present study (first100 days) indicate that after an initial activation of microglia in theaffected regions, with special regard to the peri-stroke region, theydecrease with post stroke time. However, the steepness of the de-crease is different between the ischaemic core and the peri-infarctzone and the %SUV ratios between the two regions increase withtime. This indicates that the peri-stroke region plays a critical role inthe post-stroke recovery processes, as in this region the presence ofactivated microglia can be demonstrated even several weeks afterthe insult.

In conclusion, the present clinical PET data, obtained with the TSPOmolecular imaging biomarker [

11C]vinpocetine, indicate a both tempo-

rally and regionally different microglial activation after a stroke in theaffected brain regions. Further to an initial up-regulation of microgliain and around the epicentre of the insult, there is a time-dependentsteady decrease of this activation. However, the slope of the decreaseof the microglial activation is steeper in the ischaemic core than in theperi-infarct zone, indicating a longer presence of activated microgliaand, consequently, longer lasting neuroinflammatory activity, in theperi-infarct zone.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Acknowledgements

The authors express their gratitude to the members of theKarolinska PET group for their excellent technical support, in general,and to Johan Mohlin and Julio Gabriel for their technical assistance inthe PET experiments, in particular. The study was supported partly byGedeon Richter Plc.

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