Ž .Brain Research 781 1998 1–7

Research report

Binding capacity of FK506 binding protein after 2-hour hemisphericischemia in gerbil brain

Hiroyuki Nozaki ), Kortaro Tanaka, Toshitaka Shirai, Eiichiro Nagata, Taro Kondo, Satoshi Koyama,Tomohisa Dembo, Yasuo Fukuuchi

Department of Neurology, School of Medicine, Keio UniÕersity, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan

Accepted 9 September 1997

Abstract

Ž .The binding capacity of FK506 binding protein FKBP was examined after 2-h hemispheric ischemia in the gerbil brain in order toclarify the precise mechanism of the neuroprotective effects of FK506. Firstly, the FK506 binding was evaluated in vitro in the normal

w3 xgerbil brain using 1 nM H dihydro-FK506 as a specific ligand. FK506 binding sites were distributed in a rather homogeneous manner,although the greatest binding was noted in the hippocampus CA1. Secondly, Scatchard analysis demonstrated that the binding sites ofFK506 could be composed of two components in each brain region. Thirdly, 18 Mongolian gerbils were divided into two groups: an

Ž . Ž .ischemia group ns12 and a sham group ns6 . The right common carotid artery was ligated to induce hemispheric ischemia for 2 hw14 xin the ischemia group. The local cerebral blood flow was measured at the end of the experiment by the C iodoantipyrine method. The

ligated animals with levels of local cerebral blood flow in the lateral nuclei of the thalamus of less than 50 mlr100 grmin were utilizedŽ .as the ischemia group ns6 for further data analysis. No significant differences in FK506 binding between the ischemia and sham

groups were observed in any regions. The above data indicate that the binding capacity of FKBP tends to remain normal during 2-hischemia, suggesting that FK506 may exert its neuroprotective effects through its binding to FKBP in the brain during the early phase ofcerebral ischemia. q 1998 Elsevier Science B.V.

Keywords: FK506; FK506 binding protein; Calcineurin; Cerebral ischemia; Cerebral blood flow; Gerbil

1. Introduction

FK506, which was originally developed as a new im-munosuppressant, has recently become a focus of attentiondue to its neuroprotective effects against cerebral ischemic

w xdamage 5,6,14,23 . FK506 binds to an immunophilin ofthe FK506 binding class named FK506 binding proteinŽ .FKBP , including FKBP12, FKBP52, etc. The FK506–FKBP12 complex inhibits the Ca2qrcalmodulin-dependent

w xphosphatase, calcineurin 15 , resulting in suppression ofŽ .nitric oxide synthase NOS activity, which is now consid-

ered to be one of the basic mechanisms of the neuroprotec-w xtive effects of FK506 in cerebral ischemia 2,16 .

w xLyons et al. 10 have reported an increase in FKBP12mRNA expression and FK506 binding in chronic lesionsof the peripheral nerves, indicating that FKBP12 may playan important role in neuronal regeneration. On the otherhand, despite various studies on the effects of FK506 in

) Corresponding author. Fax: q81 3 33531272; E-mail:hiroyuki_nozaki@msn.com

w xcerebral ischemia 5,6,14,23 , it has not yet been investi-gated whether the binding properties of FKBP with FK506are altered or not during cerebral ischemia. The binding ofFK506 to FKBP is an important process in the pharmaco-

w xlogical actions of this agent 4,15,17 . The distribution ofFKBP can be precisely visualized by in vitro binding

w3 x w xprocedures using H dihydro-FK506 3,16 . Recently, wefound that intracellular signal transduction via cyclicAMP-dependent protein kinase may be effectively pro-

w xtected by FK506 after 2 h of severe cerebral ischemia 22 .In addition, neuroprotective treatment at the acute stage ofstroke is now one of the most important issues in clinicalmedicine. The present study was therefore undertaken toelucidate the regional binding of FK506 in the normal andischemic gerbil brain at 2 h after onset.

2. Materials and methods

The experimental protocol described below was ap-proved as meeting the Animal Experimental Guidelines of

0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved.Ž .PII S0006-8993 97 01130-X

( )H. Nozaki et al.rBrain Research 781 1998 1–72

Keio University School of Medicine by the ExperimentalAnimal Committee.

2.1. Regional distribution and ligand binding properties ofFKBP in normal gerbil brain

2.1.1. Animal preparationSix Mongolian male gerbils weighing 60–90 g were

used. The animals were sacrificed by decapitation, andtheir brains were quickly removed and frozen by immer-

Ž .sion in liquid Freon 22 Asahi Glass, Tokyo, Japan . Eachfrozen brain was cut into consecutive coronal sections of

Ž20 mm in thickness on a cryostat Cryocut 1800, Leica.Instruments GmbH, Nussloch, Germany . Double sets of

Žsections at predetermined levels frontal cortex, caudate–putamen, globus pallidus, hippocampus, substantia nigra,

.and cerebellum from each animal were employed forw3 xcarrying out labeling procedures with H dihydro-FK506.

In addition, two sections each at the same levels werestained with cresyl violet for anatomical identification andalignment of the autoradiograms.

2.1.2. QuantitatiÕe binding studyŽSections were preincubated in buffer 50 mM HEPES

Ž .pH 7.4 , 2 mgrml bovine serum albumin, 0.02% Tween-.20 for 5 min at 258C, and subsequently preincubated in

this buffer with 5% ethanol for 60 min at 258C as de-w xscribed previously 3,16 . The sections were then incubated

w3 x Žwith 1 nM H dihydro-FK506 specific activitys100.0Cirmmol; NEN Research ProductsrDu Pont, Wilmington,

.DE, USA for 60 min at 258C. The reactions were termi-nated by two 5-min washes in ice-cold buffer. The sectionswere next rinsed in ice-cold distilled water and immedi-ately dried under a stream of cold air. The non-specificbinding was determined by a parallel incubation in thepresence of 1 mM unlabeled FK506. Autoradiograms weregenerated by apposing the slides onto 3H-sensitive filmŽ 3Ultrofilm H, Leica Instruments GmbH, Nussloch, Ger-

. 3many with calibrated H-embedded polymer standard for2 weeks at 48C. The resultant autoradiograms were ana-lyzed using an image processing system developed in ourlaboratory. All of the data presented in this report expressthe specific binding obtained by subtracting the value forthe non-specific binding from that for the total binding.

[ 3 ]2.1.3. Saturation study of H dihydro-FK506w3 xA saturation study of H dihydro-FK506 binding was

performed employing 32 sequential brain sections at eachŽof the predetermined levels caudate–putamen and hip-

.pocampus in each animal. The labeling procedure withw3 xH dihydro-FK506 was basically the same as that de-scribed above. Triplicate sections were incubated with 0.2,

w3 x0.67, 1, 4, 10, 20, 40, and 100 nM H dihydro-FK506after 60 min of preincubation. The non-specific bindingwas also evaluated by a parallel incubation in the presence

Žof an excess amount of unlabeled FK506 0.2, 0.67, 1, 4,

.10, 20, 40, and 100 mM . Autoradiograms were generatedby apposing the slides onto 3H-sensitive film for 48 h to 2weeks at 48C. The specific binding was calculated bysubtracting the non-specific binding from the total binding.

For Scatchard analysis, linear regression analysis wasperformed between the boundrfree and bound values ofthe ligand by retrieving the values of the specific bindingobtained at different ligand concentrations.

2.2. Ischemia study

2.2.1. Animal preparationEighteen male Mongolian gerbils weighing 50–100 g

Ž .were divided into two groups: an ischemia group ns12Ž . Žand a sham group ns6 . Polyethylene catheters Clay-

.Adams PE10, Becton Dickinson, Parsippany, NJ, USAwere inserted into the femoral artery and vein under lightanesthesia induced by intraperitoneal injection of pento-

Ž .barbital sodium 25 mgrkg . After the animals had com-pletely recovered from the anesthesia at 4 h after thissurgical procedure, the right common carotid artery wasdoubly ligated with silk thread through a midline cervicalincision in the ischemia group. The skin was then suturedwith silk thread. The arterial blood pressure was measured,

Ž .and arterial blood gases p O , p CO , and pH werea 2 a 2

intermittently monitored with a 168 pHrBlood Gas Ana-Ž .lyzer Corning Medical, Medfield, MA, USA to ensure

that the animals were in a normal physiological state. Thebody temperature was maintained at 37.0"0.58C through-out the experiments using a heating lamp. The sham groupunderwent the same protocol as described above exceptthat the right common carotid artery was only exposedwithout occlusion.

( )2.2.2. Measurement of local cerebral blood flow LCBFLCBF was determined by the iodoantipyrine method at

2 h after ligation of the carotid artery or sham surgery asw xdescribed elsewhere 8,12,20 . Thirteen microcuries of 4-

w14 x Žw14 xiodo-N-methyl- C antipyrine C IAP; specific activity.s54.0 mCirmmol; NEN Research ProductsrDu Pont

dissolved in 0.5 ml saline was infused at a constant rate viathe femoral venous catheter for a period of 1 min. Blood

Ž .samples 10 ml were simultaneously withdrawn from thearterial catheter every 3–5 s into capillary pipettes. At theend of the infusion period, the animals were sacrificed bydecapitation, and their brains were quickly removed and

Žfrozen by immersion in liquid Freon 22 Asahi Glass,.Tokyo, Japan . The frozen brains were placed in plastic

bags, and stored at y808C until being sectioned as de-scribed below. Timed arterial blood samples were placedon a filter paper and dried for 24 h. The materials werethen eluted for 24 h in 1 ml water and 9 ml liquid

Žscintillation cocktail ACS II, Amersham, Arlington.Heights, IL, USA . The radioactivity was determined using

Ža liquid scintillation counter LS 9800, Beckman Instru-.ments, Fullerton, CA, USA with external standardization.

( )H. Nozaki et al.rBrain Research 781 1998 1–7 3

w3 xFig. 1. Representative color-coded autoradiograms of H dihydro-FK506 binding. FK506 binding sites were diffusely distributed within the brain, but thehighest binding was noted in the hippocampus CA1.

Frozen brains were cut into sections of 20 mm inŽ .thickness on a cryostat Cryocut 1800, Leica . Double sets

Žof six brain sections, each at predetermined levels frontalcortex, caudate–putamen, globus pallidus, hippocampus,

.substantia nigra, and cerebellum , from each animal wereemployed for autoradiographic LCBF measurements and

w3 xfor labeling procedures with H dihydro-FK506. In addi-tion, two sections from each level were stained with cresylviolet for anatomical identification and alignment of theautoradiograms.

The sections assigned for LCBF measurement werethaw-mounted on gelatin-chrome alum-coated microscope

Ž .slides and dried on a hot plate 608C . The dried sectionsŽwere then placed on X-ray film SB-5, Eastman Kodak,

. w14 xRochester, NY, USA together with calibrated C em-Ž .bedded methylmethacrylate standards Amersham for 10

days. Quantitative densitometric analysis of the resultantautoradiograms was performed with a computerized digitalimage processing system developed in our laboratory as

w xdescribed previously 19,20 .

[ 3 ]2.2.3. Measurement of H dihydro-FK506 bindingThe FK506 binding was evaluated in vitro using 1 nM

w3 xH dihydro-FK506 as described above. In previous stud-w x w14 xies 18,20 , we had confirmed that C IAP was com-

pletely washed out from the brain tissue when the sections

Fig. 2. Representative graph of the saturation study. The Scatchardanalysis shown in the inserted box demonstrated that the binding sites ofFK506 may be composed at least of two components in the hippocampusCA1. Similar results were obtained for other brain regions. B, boundligand; F, free ligand.

( )H. Nozaki et al.rBrain Research 781 1998 1–74

Table 1Dissociation constant and maximal binding capacity of FK506 binding in normal gerbil brain

Ž . Ž .K nM B fmolrmgd max

High affinity Low affinity High affinity Low affinity

Caudate–putamen 0.86"0.31 16.3" 4.2 110.4"28.7 232.3"28.0Ž .Frontoparietal cortex motor area 0.70"0.12 20.8" 8.6 91.3"10.6 265.7"32.3Ž .Frontoparietal cortex sensory area 0.60"0.16 18.8" 7.6 83.6" 8.6 250.8"32.2

)Ž .Temporal cortex auditory area 0.57"0.14 21.2"11.7 79.0" 9.3 258.8"50.8) ) ††Hippocampus CA1 0.51"0.13 17.3" 7.6 87.9"10.9 321.3"42.8

) ) ) ††Hippocampus CA3 0.51"0.23 17.7" 5.0 76.2"16.5 335.1"31.4) ) †Ž .Thalamus medial nuclei 0.54"0.30 16.9" 5.5 63.4"21.9 298.1"31.1) )Ž .Thalamus lateral nuclei 0.68"0.28 19.4" 5.6 66.5"15.8 213.3"41.4

) ) ) † †† Ž .Mean"S.D.: P-0.05, P-0.01 vs. caudate–putamen; P-0.05, P-0.01 vs. thalamus lateral nuclei .

were preincubated in buffer for 5 min. No radioactivitywas detected in the brain sections on Ultrofilm 3H after

w14 xwashout of the C IAP.

2.2.4. Data analysisSince gerbils do not always display significant cerebral

w xischemia after carotid ligation 7 , we previously scored theneurological state of gerbils that had been subjected tounilateral common carotid artery occlusion, and found thatan LCBF level of 50 mlr100 grmin in the lateral nucleiof the thalamus clearly differentiated animals with severeneurological deficits including complete hemiparalysis ofthe limbs from those with mild or no apparent deficitsw x11,21 . In the present study, therefore, only those animalsthat showed LCBF levels of less than 50 mlr100 grmin inthe lateral nuclei of the thalamus were included in the

Ž .ischemia group ns6 for further data analysis. Since thelateral nuclei of the thalamus were always located in thecenter of the ischemic core in the animals with definitehemispheric ischemia, this classification clearly distin-guished animals with severe ischemia from those withmild or no apparent ischemia.

The data obtained for each group were tested statisti-Ž .cally using analysis of variance ANOVA , and the t-test

with the Bonferroni correction was performed for simulta-neous multiple comparisons when significant differenceswere noted on ANOVA. Student’s t-test was also em-ployed for statistical analysis in appropriate cases. Valuesare presented as the means"S.D.

3. Results

3.1. Regional distribution and Scatchard analysis of FK506binding sites in normal gerbil brain

w3 xRepresentative autoradiograms of the H dihydro-FK506 binding are shown in Fig. 1. These autoradiogramsrevealed that the FK506 binding sites were distributed in arather homogeneous manner within the brain, although thehighest density of binding sites was noted in the hippocam-pus CA1.

A representative graph for the saturation study is pre-sented in Fig. 2. Scatchard analysis demonstrated that thebinding sites of FK506 could be composed of two compo-nents in each brain region: one characterized by a high

Ž Ž . .affinity dissociation constant K s0.5–0.9 nM withdŽ . Žlow maximal binding capacity B 63.4–110.4max

. Žfmolrmg , and the other by a low affinity K s16.3–21.2d. Ž .nM with high B 223.3–335.1 fmolrmg . Table 1max

summarizes the data for the K and B of the high andd max

low affinity sites in each region. Neither the high affinitysites nor the low affinity sites revealed any significantdifferences in their K values among the various regions.d

On the other hand, the B of the high affinity sites in themax

caudate–putamen was higher than that in other regions,whereas the B of the low affinity sites in the hippocam-max

pus CA1 and CA3 was significantly higher than that in thecaudate–putamen and the lateral nuclei of the thalamus.

3.2. Ischemia study

3.2.1. Physiological parametersTable 2 summarizes the physiological data including the

Ž .mean arterial blood pressure MABP , p O , p CO , anda 2 a 2

pH values, which were obtained immediately before LCBFmeasurement in each group. All of these values werewithin the normal physiological ranges. None of the pa-rameters differed significantly between the groups exceptthat the p O in the ischemia group was higher than that ina 2

the sham group, due probably to hyperventilation.

Table 2Ž .Arterial blood gases and mean arterial blood pressure MABP immedi-

ately before LCBF measurement

Ž . Ž .Ischemia group ns6 Sham group ns6) )Ž .p O Torr 139.5"13.2 101.3"15.3a 2

Ž .p CO Torr 34.3"12.1 37.5"2.0a 2

pH 7.37"0.27 7.27"0.07Ž .MABP mmHg 70.8"14.7 8.0"17.3

Mean"S.D.; ) ) P -0.01 vs. sham group.

( )H. Nozaki et al.rBrain Research 781 1998 1–7 5

Fig. 3. Representative color-coded autoradiograms at the level of the hippocampus. The left column shows color-coded LCBF autoradiograms, and thew3 xright column shows color-coded H dihydro-FK506 autoradiograms. The upper panel was obtained from the sham group, and the lower panel was

obtained from the ischemia group. The viewer’s right is the right side of the brain.

Table 3LCBF and FK506 binding

Ž . Ž .LCBF mlr100 grmin FK506 binding fmolrmg

Ž . Ž . Ž . Ž .Ischemia group ns6 Sham group ns6 Ischemia group ns6 Sham group ns6) ) ††Frontal cortex right 8.8" 9.0 84.7"18.4 56.2" 7.6 52.8" 7.8)left 57.0"11.9 85.3"18.4 56.6" 7.1 52.9" 7.3) ) ††Ž .Frontoparietal cortex motor area right 10.8"11.5 105.8"29.7 58.9"13.0 62.6" 7.0)left 66.7"13.0 108.5"29.2 59.2"12.7 62.8" 8.0) ) ††Ž .Frontoparietal cortex sensory area right 1.5" 2.5 110.0"36.2 58.4"13.1 59.5" 7.6

left 94.0"29.1 112.7"33.2 59.1"10.5 60.0" 8.1) ) ††Ž .Temporal cortex auditory area right 1.0" 2.4 118.2"39.2 61.9"13.9 63.5" 3.7

left 77.5"26.5 117.7"35.2 61.6"13.3 62.9" 3.8) ) ††Caudate–putamen right 4.3" 4.8 89.2"16.4 69.6"10.7 76.9" 5.0

left 58.7"19.1 93.0"21.0 69.7"11.5 75.1" 5.2) ) ††Hippocampus CA1 right 5.0" 5.5 86.8"23.4 87.7"11.1 87.3"10.9

left 59.0"10.3 89.0"27.7 86.3"10.5 88.0"12.0) ) ††Hippocampus CA3 right 5.5" 6.1 87.5"23.7 69.4"15.2 75.3"13.4

left 60.2"11.4 92.3"24.5 68.6"14.5 74.7"12.6)Ž .Thalamus medial nuclei right 47.2"22.6 115.7"32.4 58.2"11.3 68.4"10.5)left 71.0"12.0 118.2"31.7 57.8"11.7 68.8"11.4) ) ††Ž .Thalamus lateral nuclei right 3.0" 3.5 106.7"31.7 50.4"11.0 53.0" 6.8)left 60.5" 7.8 111.5"20.6 48.7" 9.9 53.0" 8.3

Cerebellum right 75.7"12.4 117.7"33.9 45.2" 8.2 50.6"10.4left 75.3"11.0 116.0"34.5 44.8" 7.4 50.2"10.1

Mean"S.D.: ) ) P-0.01, ) P-0.05 vs. sham group; †† P-0.01 vs. left side.

( )H. Nozaki et al.rBrain Research 781 1998 1–76

3.2.2. Local cerebral blood flowRepresentative color-coded LCBF autoradiograms at the

level of the hippocampus are shown in Fig. 3. As expected,a significant reduction of LCBF was noted in the righthemisphere of the animals in the ischemia group except for

Ž .the midline structures medial nuclei of the thalamus, etc.which demonstrated a low residual flow. The results of theLCBF measurements are summarized in Table 3. Eachregion of the right hemisphere in the ischemia groupexhibited a markedly reduced LCBF value, which was

Ž .significantly lower p-0.01 than that in the sham group.The anterior cortices including the frontal and frontopari-etal cortices, and the deep structures including the cau-date–putamen, lateral nuclei of the thalamus, and hip-

Ž .pocampus CA1 and CA3 regions , revealed LCBF valuesthat were less than 15% of those in the sham group. Eachregion of the left hemisphere in the ischemia group exhib-ited a moderate reduction in LCBF, some values being

Ž .significantly lower P-0.05 than those in the shamgroup. The LCBF of the cerebellar cortices on both sidesin the ischemia group revealed a mild reduction as com-pared to the sham group.

3.2.3. FK506 bindingFig. 3 shows representative color-coded autoradiograms

w3 xof the H dihydro-FK506 binding at the level of thehippocampus. No significant alteration of FK506 bindingwas seen in any region in the ischemia group as comparedto the sham group. The data for the FK506 binding aresummarized in Table 3. Comparisons between the is-chemia and sham groups demonstrated no significant dif-ferences in any region.

4. Discussion

Ž .The present experimental results suggested that aFKBP is distributed in a rather homogeneous manner inthe gerbil brain, although the most dense distribution may

Ž .exist in the hippocampus CA1, b the binding sites ofFK506 are composed of at least two components in the

Ž .gerbil brain, and c the binding capacity of FKBP remainsnormal during 2-h cerebral ischemia.

Various forms of FKBP are present not only in thew xmembrane fraction, but also in the cytosolic fraction 3,16 .

Since our incubation protocol is thought to wash outsoluble proteins from the unfixed brain tissue into thebuffer solution, the present study is considered to demon-strate mainly the membrane-bound FKBP. In fact, Dawson

w x w3 xet al. 3 have reported that the distribution of H dihydro-FK506 binding sites parallels that of FKBP in the mem-brane fraction rather than in the cytosolic fraction of therat brain. The distribution pattern of FK506 binding sitesobtained in the present study is compatible with that in the

w xrat brain 3 , suggesting that FKBP is distributed in asimilar manner in both the gerbil and rat brains.

A previous saturation study performed on the rat brainw x16 revealed two components of FK506 binding sites,

Ž .including high-affinity binding sites K s0.6 nM anddŽ .low-affinity binding sites K s30 nM . These values ared

compatible with the present data, indicating that FKBPexhibits similar binding kinetics in both the gerbil and ratbrains. Among the various forms of FKBP, FKBP12

Ždemonstrates a high-affinity binding property K s0.4d. w xnM 1,13 , while FKBP25 exhibits a low-affinity binding

w xproperty 15 .Based on the B values obtained in the present study,max

the high affinity component may be most abundantlylocated in the caudate–putamen, whereas the low-affinitycomponent may be most concentrated in the hippocampusCA3 and CA1. We assume therefore that the two bindingcomponents observed in the present study may derive fromat least two different forms of FKBP. The high-affinitycomponent may represent FKBP12, since the K values ofd

FKBP12 and the high-affinity component are very similar.w3 xIn the ischemia study, we employed 1 nM H dihydro-

FK506 as the concentration of the ligand, so that theFK506 binding obtained may mainly reflect the high-affin-ity binding component of FKBP, such as FKBP12. Takentogether, the present data indicated that the binding proper-ties of FKBP12 may remain stable within the gerbil brainin spite of 2-h ischemia.

Recently, injury of the peripheral nerves has been re-ported to enhance the expression of FKBP12 at 7–14 days

w xafter the injury 10 , suggesting that FKBP12 may play animportant role in neuronal regeneration at the chronicstage. In our preliminary study, we found no significantchanges in FK506 binding after 6 h of cerebral ischemia.Although the aim of the present experiments was to eluci-date the precise mechanism underlying the acute protectiveeffects of FK506, we also need to examine the FK506binding and expression of FKBP at the more chronic phaseof cerebral ischemia.

FK506 has recently been shown to exert protectivew xactions in cerebral ischemia 5,6,14,23 , and such effects

may be closely associated with the inhibitory action ofw xFK506–FKBP12 complex upon calcineurin 2,16 . Inhibi-

tion of calcineurin is expected to enhance the phosphoryla-tion state of NOS and other proteins such as growth-asso-

Ž .ciated protein 43 GAP43 . Phosphorylation of GAP43augmented by FK506 has been reported to enhance the

w xextension of neuronal processes 9 . A recent report byw xSteiner et al. 17 , however, has suggested that the inhibi-

tion of rotamase activity of FKBP induced by binding ofFK506 to FKBP may be responsible for the neurotrophiceffects of this agent. Nevertheless, a preserved bindingcapacity of FKBP with FK506 may be an essential pre-requisite for manifestation of the various protective effectsof FK506 in cerebral ischemia. The present findings indi-cate therefore that FK506 may bind to FKBP and formFK506-FKBP complex, resulting in the expression of neu-roprotective effects after 2 h of cerebral ischemia. The

( )H. Nozaki et al.rBrain Research 781 1998 1–7 7

rather homogeneous distribution of FK506 binding withinthe gerbil brain appears to ensure that FK506 can exert itsprotective effects extensively throughout the gerbil brain.

Acknowledgements

Ž .FK506 tacrolimus was donated by Fujisawa Pharma-ceutical Co., Ltd., Osaka, Japan. The authors would like to

Ž .thank Dr. Shintaro Gomi Aoyama Gakuin University andŽDr. Ban Mihara Department of Neurology, Mihara

.Memorial Hospital for their helpful advice.

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