Protection Against Ischemic Brain Damagein Rats by Immunophilin Ligand GPI-1046

Feng Li, Nobuhiko Omori, Takeshi Hayashi, Guang Jin, Keiko Sato, Isao Nagano,Mikio Shoji, and Koji Abe*Department of Neurology, Graduate School of Medicine and Dentistry, Okayama University, Okayama, Japan

To determine the effect of immunophilin ligand GPI-1046on ischemic brain injury, 90 min of transient middle ce-rebral artery occlusion (MCAO) was carried out in ratbrains. In contrast to cases treated with vehicle, theinfarct volume was reduced greatly and rotamase activitywas inhibited significantly at 24 hr of reperfusion bytreatment with GPI-1046. Immunoreactivity and the num-ber of cells stained positively for FKBP12, FKBP52,caspase-8, cytochrome c, and caspase-3 were also re-duced markedly in the brain after GPI-1046 treatment.The present results suggest that GPI-1046 significantlydecreased infarct volume and provided neuroprotectiveeffect on rats after transient focal cerebral ischemia byinhibiting the increase of rotamase activity and of thenumber of FKBP12-, FKBP52-, caspase-8-, cytochromec-, and caspase-3-positive cells in the ischemic area.© 2004 Wiley-Liss, Inc.

Key words: caspase; FKBPs; GPI-1046; rotamase

Immunophilin ligand GPI-1046 is an analogue ofimmunosuppressant tacrolimus (FK506), but does nothave immunosuppressive properties. GPI-1046 exertsneuroprotective effect by binding a distinct receptor pro-tein termed immunophilin FKBPs (FK506 binding pro-teins). Although the neuroprotective roles of GPI-1046 inneuronal injury remain controversial, several studies haveindicated that GPI-1046 represents a very potent activityof neuroprotection in animal models of neurodegenera-tion and on neurite outgrowth in cultured chick dorsalroot ganglia cells (Steiner et al., 1997; Parker et al., 2000;Khan et al., 2002; Moss et al., 2002). Steiner et al. (1997)reported that GPI-1046 promotes protection and sprout-ing of serotonin-containing nerve fibers in somatosensorycerebral cortex after parachloroamphetamine treatment,and stimulates the regeneration of lesioned sciatic nerveaxons and myelin levels in vivo.

FKBPs belong to the large family of peptidyl-prolylcis-trans isomerases (rotamase activity) that is inhibited byimmunophilin ligand binding (Marks, 1996). FKBPs thusare involved in many cellular processes such as cell signal-ing, protein trafficking, and transcription (Harrar et al.,2001). Binding the immunophilins FKBP12 and FKBP52(FK506 binding protein of 12 kDa or 52 kDa, respec-tively) has been proposed to mediate the neurotrophic

effects of GPI-1046 (Hamilton and Steiner, 1998; Gold etal., 1999). FKBP12 is expressed primarily in neurons, andthe levels increase markedly after cerebral ischemia, sciaticnerve crush injury, and Parkinson’s disease (Lyons et al.,1995; Kato et al., 2000). On the other hand, FKBP52 (alsoknown as FKBP59 or heat shock protein 56) functions asa molecular chaperone and is enhanced in the substantianigra of Parkinson’s patients’ brains (Avramut and Achim,2002).

A recent study has demonstrated that enforceddimerization of FKBP and the Fas-associated death do-main protein (FADD) by the bivalent ligand FK1012resulted in activation of caspase-dependent apoptotic sig-nals and caspase-independent necrotic death signals (Mat-sumura et al., 2000). FADD-mediated death signalingrequires the activation of a class of cysteine proteases, andplays an important role in cerebral ischemic death (Jin etal., 2001). Caspase-8 possesses a sequence homologous tothe death effector domain (DED) of FADD within itspro-caspase-8, contains a protease domain, and is capableof cleaving caspase-3 substrates (Srinivasula et al., 1996).Matsushita et al. (2000) reported that transient spinal cordischemia induces the formation of a death-inducing sig-naling complex (including Fas, FADD, and caspase-8), andparticipates in caspase-8 activation and sequentialcaspase-3 cleavage. Active caspase-8 initiates downstreamcleavage of caspase-3 by direct or mitochondrial-

Abbreviations: CBF, cerebral blood flow; DED, death effector domain;DMSO, dimethyl sulfoxide; FADD, Fas-associated death domain protein;FKBPs, FK506 binding proteins; MCA, middle cerebral artery; MCAO,middle cerebral artery occlusion; PBS, phosphate-buffered saline; SC, shamcontrol; TTC, 2,3,5-triphenyltetrazolium chloride.

Contract grant sponsor: Ministry of Education, Science, Culture and Sportsof Japan; Contract grant number: 12470141, 15659338; Contract grantsponsor: Ministry of Health and Welfare of Japan.

*Correspondence to: Prof. Koji Abe, MD, PhD, Department of Neurol-ogy, Graduate School of Medicine and Dentistry, Okayama University,2-5-1 Shikatacho, Okayama 700-8558 Japan.E-mail: lifeng@cc.okayama-u.ac.jp

Received 17 October 2003; Revised 19 December 2003; Accepted 30December 2003

Published online 23 March 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jnr.20067

Journal of Neuroscience Research 76:383–389 (2004)

© 2004 Wiley-Liss, Inc.

dependent cytochrome c release, leading to apoptosis (Ku-wana et al., 1998; Matsushita et al., 2000).

Neuronal cell death resulting from cerebral ischemiainvolves both necrotic and apoptotic mechanisms (Abe etal., 1995; Abe, 1999). Necrotic cell death occurs rapidly inthe ischemic core region and apoptotic neuronal damagedevelops more slowly in the peri-infarct area (ischemicpenumbra) after middle cerebral artery occlusion(MCAO) (Furlan et al., 1996; Abe, 2000). Although FK-BPs may be fundamentally involved in such neuronaldeath, a possible neuroprotective effect of immunophilinligand GPI-1046 has not been examined previously intransient focal cerebral ischemia, and the mechanism of theputative protective effect against ischemic injury remains amystery. We therefore investigated a possible protectiveeffect of GPI-1046 on infarct size in association with theinhibition of rotamase activity and FKBP or caspase im-munoreactivity after transient MCAO in rats.

MATERIALS AND METHODS

Animal Model

Adult male Wistar rats (body weight 250–280 g) wereanesthetized with an intraperitoneal (i.p.) injection of pentobar-bital (10 mg/250 g rat), and a burr hole with a diameter of 2 mmfor measuring regional cerebral blood flow (rCBF) was madecarefully in the skull using an electric dental drill to avoidtraumatic brain injury. The location of the burr hole was 3 mmdorsal and 4 mm lateral to the right from the bregma, which islocated in the upper part of middle cerebral artery (MCA)territory. Dura mater was preserved at this time. The animalswere allowed to recover under ambient conditions.

On the next day, about 24 hr after the drilling, the ratswere anesthetized by inhalation of a nitrous oxide-oxygen-halothane (69:30:1%) mixture during surgical preparation. Theright MCA was occluded by insertion of a silicon-coated nylonthread through the common carotid artery as described previ-ously (Kitagawa et al., 1998). Body temperature was maintainedat 37 � 0.3°C during surgery for MCA occlusion (MCAO).After 90 min of transient MCAO, cerebral blood flow (CBF)was restored by withdrawal of the nylon thread. Sham control(SC) animals were treated in the same way without MCAO(n � 7). Treatment group (n � 21) received injections ofGPI-1046 (30 mg/kg, subcutaneously [s.c.]) 2 hr before MCAOand immediately after the reperfusion. This subcutaneous dose,selected according to results of previous studies, remains potent

and keeps a continuous concentration of GPI-1046 in rat brain(Steiner et al., 1997; Parker et al., 2000). GPI-1046 was dis-solved (80 mg/ml) in 1.0% dimethyl sulfoxide (DMSO). Thecontrol group (n � 21) received injections of vehicle only (1.0%DMSO). Blood samples (90 �l) were collected before, duringthe 90 min MCAO, and just after the reperfusion from the tailartery for measurement of pH, PO2, and PCO2 (blood gasanalyzer Model ABL330; Radiometer). Regional CBF (rCBF)of the right frontoparietal cortex was measured using LaserDoppler flowmetry (FLO-C1; Omega Flow) before, during,and just after reperfusion. Blood pressure was also measuredusing a blood pressure monitor (MK-1030; Muromachi KikaiCo., Ltd.). Animals were allowed to recover at ambient tem-perature (21–24°C) until sampling at 24 hr after reperfusion.Experimental protocol and procedures were approved by theAnimal Committee of Okayama University, Japan.

Estimation of Infarct Volume After Transient MCAO

To examine a possible effect of GPI-1046 on infarct sizeafter transient MCAO, rat forebrains were removed and dividedinto five coronal (2-mm) sections 24 hr after reperfusion withvehicle or GPI-1046 (n � 7 for each group) treatment. Coronalsections were stained with saline containing 2% 2,3,5-triphenyltetrazolium chloride (TTC) at 37°C for 30 min, afterwhich sections were fixed in 10% neutralized formalin as de-scribed previously (Kitagawa et al., 1998). The infarct area ofeach section was measured by UTHSCSA Image Tool v. 3.00software, and then infarct areas on each slice were summed andmultiplied by slice thickness to give the infarct volume.

Preparation of Cytoplasmic Extracts and RotamaseActivity Analysis

The animals were decapitated 24 hr after reperfusion (n �7 for each of sham control, vehicle- and GPI-1046-treatedgroups). Intracardiac perfusion with heparinized 0.9% saline wascarried out before brain extraction to eliminate erythrocytes

TABLE I. Physiologic Parameters in Middle Cerebral Artery Occluded Rats*

Parameter

Vehicle GPI-1046

MCAO Restoration MCAO Restoration

Pre Post Pre Post Pre Post Pre Post

pH 7.44 � 0.02 7.45 � 0.01 7.40 � 0.03 7.42 � 0.02 7.45 � 0.02 7.43 � 0.01 7.43 � 0.02 7.44 � 0.01PO2 (mmHg) 112.2 � 12.1 120.4 � 14.8 118.1 � 15.0 119.8 � 13.0 117.6 � 11.5 114.9 � 13.2 114.8 � 12.0 116.3 � 11.4PCO2 (mmHg) 41.2 � 4.3 42.8 � 4.2 42.4 � 5.0 40.9 � 3.8 39.8 � 3.6 41.5 � 4.7 42.0 � 4.1 41.1 � 3.9Rectal temp (°C) 37.1 � 0.1 36.8 � 0.3 36.7 � 0.2 37.0 � 0.2 37.1 � 0.1 36.9 � 0.2 36.8 � 0.2 37.0 � 0.1rCBF 100 17.7 � 5.6 14.9 � 4.1 91.5 � 7.3 100 16.8 � 6.2 14.4 � 3.7 92.0 � 6.9

*Data are expressed as mean � standard deviation. MCAO, middle cerebral artery occlusion; temp, temperature; rCBF, regional cerebral blood flow.

TABLE II. Rectal Temperature after Reperfusion*

Treatment

Time after reperfusion (hr)

1 2 6 24

Vehicle 36.9 � 0.2 37.0 � 0.1 36.8 � 0.3 37.1 � 0.1GPI-1046 37.1 � 0.1 36.7 � 0.2 36.9 � 0.1 37.0 � 0.2

*Data are expressed as mean temperature (°C) � standard deviation.

384 Li et al.

from the tissue. The cerebral cortex of the right MCAO areawas removed and homogenized gently in a glass tissue grinder indouble weight volume of 1 M Tris-HCl solution (pH 7.4). Thehomogenate was centrifuged at 4°C at 60,000 � g for 15 min(TL-100; Beckman Automatic Preparative Ultracentrifuge,Central Research Laboratory of Medical School of OkayamaUniversity, Japan), and the supernatant was used for rotamaseactivity analysis.

The measurement of rotamase activity was based on amethod described previously (Fischer et al., 1989). Briefly, thecis-trans isomerization of an alanine-proline bond in a modelsubstrate, N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (a testpeptide; Sigma, St. Louis, MO), was monitored spectrophoto-metrically in a chymotrypsin-coupled assay, which releases para-nitroanilide from the trans form of the substrate peptide. Underequilibrium conditions, about 85% of the peptide was in thetrans form and was cleaved readily by �-chymotrypsin (Sigma).The remaining peptide, having a cis conformation, was cleavedupon enzymatic conversion to the trans form. The peptide wasdissolved in ethanol to a final concentration of 7.5 mM. An assaysolution (850 �l of 35 mM HEPES, pH 7.8; 10 �l of 7.5 mMpeptide; and 100 �l of �-chymotrypsin) was added to cuvette at4°C on the UV spectrophotometer (UV-1200; Shimadzu Cor-poration, Kyoto, Japan) at 390 nm. The trans isomer of thesubstrate was consumed by �-chymotrypsin hydrolysis duringthe mixing time. The absorbance of the sample was immediatelyrecorded. This corresponds to the amount of the trans isomerpresent in the original mixture. After 30 sec, 40 �l of theprepared sample supernatant was added to increase the rate ofcis-to-trans isomerization, and the absorbance was measured afterthe �-chymotrypsin-catalyzed hydrolysis was complete (1 min).The difference between the initial and the final absorbance canreflect rotamase activity.

Brain Sample Preparation and Immunohistochemistry

At 24 hr after the reperfusion, rat forebrains were removedwith deep anesthesia and quickly frozen for each experimentalgroup (n � 7, for each of vehicle- and GPI-1046-treatedgroups). SC sections (n � 7) were also obtained in the samefashion. Coronal sections at the caudate level were cut on acryostat at �18°C to a 10-�m thickness and collected on glassslides coated with poly-L-lysine. For immunohistochemistryanalysis, coronal sections were fixed in 4% paraformaldehyde inphosphate-buffered saline (PBS) for 15 min, and were thenincubated, first with rabbit FKBP12 or FKBP52 antibody (1:100dilution, PA1-026 and PA3-020, respectively; Affinity BioRe-agents), mouse caspase-8 p20 (D-8) or rabbit cytochrome c(H-104) antibody (1:50 dilution, sc-5263 and sc-7159, respec-tively; Santa Cruz Biotechnology, Santa Cruz, CA), and rabbitcleaved caspase-3 (Asp175) antibody (1:25 dilution, #9661; CellSignaling Tech) overnight at 4°C. Some sections were treatedsimultaneously without the first antibody. Specificity of the firstantibodies has been described elsewhere (Czar et al., 1994; Huet al., 2000; Jin et al., 2001; Noshita et al., 2001). After washing,sections were incubated for 2 hr with biotinylated secondaryantibodies, a 1:200 dilution of goat anti-rabbit IgG (immuno-globulin G) and horse anti-mouse (H�L) [horse anti-mouse(heavy � light)] (Vector Laboratories), followed by incubationfor 30 min with avidin-biotin-horseradish peroxidase complex,and finally developed with diaminobenzidine tetrahydrochlo-ride. Sections were examined using a light microscope, andstained cells in 1 mm2 of three MCA areas were counted,summed, and averaged in the ischemic core and in the peri-infarct area (ischemic penumbra).

Statistical Analysis

Statistical analyses were carried out using one-way analysisof variance (ANOVA) followed by Bonferroni test for rotamase

Fig. 1. Effects of GPI-1046 on infarct volume at 24 hr of reperfusionafter 90 min of transient MCAO. Infarct volume in the brain sectionwas reduced significantly by GPI-1046 treatment (*P � 0.022).

Fig. 2. Inhibition of rotamase activation by GPI-1046. Rotamase ac-tivity (UV absorbance) was increased significantly at 24 hr of reperfu-sion after 90 min of transient MCAO (**P � 0.001), which is reducedmarkedly by GPI-1046 treatment (*P � 0.05).

Neuroprotection of GPI-1046 in MCAO 385

Figure 3

activity or the number of staining cells, paired t-test (two-tailed)for infarct volume, and ANOVA repeated measure for physio-logic or rCBF data, respectively. Values were considered to besignificant when P � 0.05. Data are presented as mean �standard deviation (SD).

RESULTSEffect of GPI-1046 on Infarct Volume

Physiologic parameters are shown in Table I andTable II. There was no significant difference in physio-logic parameters such as pH, PO2, and PCO2 between thevehicle- and GPI-1046-treated groups before MCAO,during MCAO, or after reperfusion (Table I). There wasno significant difference in rCBF between the vehicle andGPI-1046 groups (Table I). There was also no significantdifference in rectal temperature at 1, 2, 6, and 24 hr afterreperfusion between the vehicle- and GPI-1046-treatedgroups (Table II). The infarct volume of the GPI-1046-treated group was 97.5 � 46.6 mm3 (mean � SD; n � 7),which was significantly smaller (P � 0.022) than thatof the vehicle-treated group (335.5 � 73.6 mm3; n � 7)(Fig. 1).

Effect of GPI-1046 on Rotamase ActivityInhibition

Assessment of rotamase activity revealed potent in-hibition by GPI-1046 (Fig. 2). The absorbance values ofthe vehicle-treated group were increased significantly(mean � SD, 0.240 � 0.047; n � 7, P � 0.0002) ascompared to the SC group (0.137 � 0.023; n � 7). Onthe other hand, the absorbance values of GPI-1046-treatedgroup were reduced markedly (0.173 � 0.040; n � 7, P �0.0135) compared to the vehicle group.

Immunohistochemical Study of FKBP12, FKBP52,Caspase-8, Cytochrome c, and Caspase-3

Results of the immunohistochemical study forFKBP12, FKBP52, caspase-8, cytochrome c, andcaspase-3 are summarized in Figures 3 and 4 and Table III.In all stainings, there were no positive cells in the samplewithout first antibody as a negative control (data notshown). Immunoreactivity for FKBP12 (Fig. 3a,b) andFKBP52 (Fig. 3g,h) was weakly detectable in SC brains(n � 7 for each); however, FKBP12 and FKBP52 becamestrongly stained at 24 hr after reperfusion in the penumbra(Fig. 3d and 3j, respectively), but it disappeared in theischemic core (Fig.3c and 3i, respectively). With GPI-1046 treatment, the number of stained cells for FKBP12

and FKBP52 was reduced significantly in the ischemicpenumbra (Fig. 3f and 3l, respectively), but not in theischemic core (Fig. 3e and 3k, respectively).

Caspase-8 (Fig. 3m,n), cytochrome c (Fig. 3s,t), andcaspase-3 (Fig. 3y,z) staining were negative in SC brains(n � 7 for each). Strong immunoreactivity for caspase-8was induced in the ischemic penumbra (Fig. 3p), but notin the ischemic core (Fig. 3o) 24 hr after reperfusion.Cytochrome c staining increased significantly in both theischemic core and penumbra (Fig. 3u,v). Immunoreactivecaspase-3 staining was induced strongly in the ischemicpenumbra (Fig. 3B), but positive cells were not as numer-ous as those were for caspase-8 (Fig. 4). Caspase-3 was notinduced in the ischemic core in the vehicle-treated tissues(Fig. 3A). Treatment with GPI-1046 significantly reducedthe number of positive cells in the ischemic penumbra(Fig. 3r, 3x, and 3D, respectively), but not in the ischemiccore (Fig. 3q, 3w, and 3C, respectively), as compared tothat in the vehicle-treated tissues.

The results of the quantitative analysis for all stainingsare shown in Figure 4 and Table III. The number ofFKBP12- and FKBP52-positive cells was enhanced signif-icantly in the vehicle-treated group (P � 0.001), but notin the GPI-1046-treated group (P 0.05), as compared tothe SC group. The reduction of these positive cells withGPI-1046 treatment was statistically significant as com-pared to vehicle treatment (P � 0.001). For caspase-8,cytochrome c, and caspase-3, the number of positivelystained cells was also reduced markedly by GPI-1046treatment (P � 0.001).

Š

Fig. 3. The topographical distribution of the ischemic damage is shownat bottom as a schematic. Coronal section of the rat brain is at the levelof bregma �0.26 mm based on the rat brain atlas of Paxinos andWatson. Ischemic core and peri-infarct area (ischemic penumbra) forimmunohistochemical analyses are illustrated. Representative photomi-crographs of FKBP12 (a–f), FKBP52 (g–l), caspase-8 (m–r), cyto-chrome c (s–x) and caspase-3 (y–D) in the ischemic core (core) andperi-infarct area (penumbra) of vehicle treatment (c, d, i, j, o, p, u, v,

A, B), and GPI-1046 treatment (e, f, k, l, q, r, w, x, C, D), with shamcontrol (SC; a, b, g, h, m, n, s, t, y, z). Note strong induction of thesepositive cells with vehicle treatment (d, j, p, v, B), and its significantreduction with GPI-1046 treatment (f, l, r, x, D) in the ischemicpenumbra. Also note similar strong inductions of cytochrome c in theischemic core of vehicle (u) and GPI-1046 (w) treatments. Scale bar(D) � 100 �m. Figure can be viewed in color online via www.inter-science.wiley.com.

Fig. 4. The number of FKBP12-, FKBP52-, caspase-8-, cytochromec-, and caspase-3-positive cells in the peri-infarct area (penumbra) ofischemic brain. Note marked increase of FKBP12- and FKBP52-positive cells in vehicle treatment (*P � 0.001), and significant reduc-tion of these positive cells in GPI-1046 treatment (*P � 0.001).

Neuroprotection of GPI-1046 in MCAO 387

DISCUSSIONThe important finding of the present study is that the

immunophilin ligand GPI-1046 decreased infarct volumeand provided neuroprotective effect in rats after transientfocal cerebral ischemia (Fig. 1). GPI-1046 inhibited theincrease of rotamase activity (Fig. 2) and the increase inFKBP12-, FKBP52-, caspase-8-, cytochrome c-, andcaspase-3-positive cells in the ischemic area (Fig. 3, 4, andTable III).

Many studies have shown that GPI-1046 exerts neu-roprotection in vivo and in vitro (Steiner et al., 1997),improves spatial memory and reverses cholinergic fiberatrophy in aged mice (Sauer et al., 1999). Guo et al.(2001a) reported that GPI-1046 provided a neuroprotec-tive effect by inhibiting rotamase activity in 1-methyl-4-phenylpyr-idinium (MPP�) and 6-hydroxydopamine (6-OHDA) toxicity models. Parker et al. (2000) reported thatGPI-1046 did not have a neuroprotective effect when ratswere treated with GPI-1046 10 min after permanentMCAO (3 mg/kg intravenously or 10–30 mg/kg s.c.),and quantification of infarct volume was carried out at24 hr after MCAO. The present study, however, em-ployed GPI-1046 at 2 hr before the onset of MCAO andimmediately after reperfusion, and showed that the neu-roprotective effect of GPI-1046 was associated with inhi-bition of rotamase activity and caspase activation (Fig.2–4). Because our preliminary study revealed that onlypre- or post-ischemic GPI-1046 application showed trivialneuroprotective effect (data not shown), the present ex-periment employed both pre- and post-ischemic drugapplication.

Matsumura et al. (2000) demonstrated that FKBP-FADD results in both caspase-dependent apoptotic signalsand caspase-independent necrotic death signals in vitro. Jinet al. (2001) have demonstrated that FADD is upregulatedand activates caspase-10 through the DED of FADD, andthen results subsequently in activation of downstreamcomponents such as caspase-3 after global cerebral isch-emia. The level of FKBP12 is elevated in cerebral ischemiaand FKBP52 is elevated in Parkinson’s patients’ brains(Kato et al., 2000; Avramut and Achim, 2002). Our resultshave confirmed that FKBP12, FKBP52, and caspase-8were activated markedly in the peri-infarct area (ischemicpenumbra) after transient MCAO, but it remains unclearhow FKBPs interact with FADD under physiologic or

ischemic conditions. It seems likely that activated FKBPsinteract more easily with FADD and trigger FADD acti-vation, contributing to neuronal apoptosis as well as Fasafter ischemic insults. Further study will be required tounderstand the relationship between FKBPs and FADD inischemic brain injury.

FKBPs have dual roles as rotamases and chaperones,and rotamase activity is inhibited by immunophilin ligands(Snyder and Lai, 1998; Guo et al., 2001a). Apoptoticmechanisms are activated during ischemia and that inhi-bition of apoptosis reduces ischemic brain damage (Kita-gawa and Abe, 1998; Abe, 2000; Xu et al., 2003). Becauseour results have showed that GPI-1046 significantly in-hibited rotamase activity and reduced FKBP12, FKBP52,and caspase-8 activations (Figs. 2–4), the neuroprotectiveaction of GPI-1046 may be attributable to its inhibition ofrotamase activity and FKBPs. Chaperone activity of FK-BPs was reduced by GPI-1046, which is likely to opposecaspase-independent necrotic death resulting from FKBP-FADD activation (Chambraud et al., 1999; Guo et al.,2001b).

The infarct after transient MCAO completes rapidlyby the necrotic mechanism in the ischemic core, whereasthe neural cells of the ischemic penumbral regions moreslowly die by apoptotic mechanisms (Mattson et al., 2000;Xu et al., 2003). Because GPI-1046 significantly inhibitscytochrome c and caspase-3 immunoreactivity and stainedcell numbers in ischemic penumbra (Fig. 3 and 4), GPI-1046-mediated protection against cerebral ischemia in-volves antiapoptotic mechanisms. On the other hand, inthe ischemic core, FKBP12 or FKBP52 disappeared andcaspase-8, cytochrome c and caspase-3 were as the case ofvehicle-treated group (Fig. 3). Thus, GPI-1046 may fail toexhibit a protective effect on neurons of the ischemic corebecause of rapid necrotic neuronal death resulted fromstrong ischemic insults in the core.

In conclusion, the present study demonstrated theneuroprotective effect of GPI-1046 on ischemic braininjury induced by transient MCAO, which was associ-ated strongly with inhibition of rotamase activity andreduction of apoptotic processes. GPI-1046 thereforecould become a strong candidate as a therapeutic agentin the treatment of ischemic brain disease of humanstroke patients.

TABLE III. Number of Cells Stained for FKBP12, FKBP52, Caspase-8, Cytochrome c, and Caspase-3*

Sham control Vehicle GPI-1046

Core Penumbra Core Penumbra Core Penumbra

FKBP12 110.3 � 19.8 111.6 � 16.4 0 338.6 � 66.6 0 162.4 � 10.0FKBP52 118.1 � 15.5 114.0 � 10.4 0 265.0 � 47.9 0 153.4 � 11.3Cas-8 0 0 0 396.1 � 82.1 0 128.6 � 50.8Cyt c 0 0 339.6 � 47.4 352.9 � 27.9 355.6 � 51.7 167.9 � 23.5Cas-3 0 0 0 125.9 � 46.6 0 20.9 � 9.0

*Number of cells per mm2 stained for FK506 binding protein (FKBP)12, FKBP52, caspase-8 (cas-8), cytochrome c (cyt c), and caspase-3 (cas-3). Dataare expressed as mean � standard deviation.

388 Li et al.

REFERENCESAbe K. 1999. Neurons: necrotic vs. apoptotic changes. Cereb Ischemia

1:217–232.Abe K. 2000. Therapeutic potential of neurotrophic factors and neural stem

cells against ischemic brain injury. J Cereb Blood Flow Metab 20:1393–1408.

Abe K, Aoki K, Kawagoe J, Yoshida T, Hattori A, Kogure K, Itoyama Y.1995. Ischemic delayed neuronal death: a mitochondrial hypothesis.Stroke 26:1478–1489.

Avramut M, Achim CL. 2002. Immunophilins and their ligands: insightsinto survival and growth of human neurons. Physiol Behav 77:463–468.

Chambraud B, Radanyi C, Camonis JH, Rajkowski K, Schumacher M,Baulieu EE. 1999. Immunophilins, Refsum disease, and lupus nephritis:the peroxisomal enzyme phytanoyl-COA�-hydroxylase is a new FKBP-associated protein. Proc Natl Acad Sci USA 96:2104–2109.

Czar MJ, Owens-Grillo JK, Yem AW, Leach KL, Deibel Jr MR, WelshMJ, Pratt WB. 1994. The hsp56 immunophilin component of untrans-formed steroid receptor complexes is localized both to microtubules in thecytoplasm and to the same nonrandom regions within the nucleus as thesteroid receptor. Mol Endocrinol 8:1731–1741.

Fischer G, Wittmann-Liebold B, Lang K, Kiefhaber T, Schmid FX. 1989.Cyclophilin and peptidyl-prolyl cis-trans isomerase are probably identicalproteins. Nature 340:351–352.

Furlan M, Marchal G, Viader F, Derlon JM, Baron JC. 1996. Spontaneousneurological recovery after stroke and the fate of the ischemic penumbra.Ann Neurol 40:216–226.

Gold BG, Densmore V, Shou W, Matzuk MM, Gordon HS. 1999.Immunophilin FK506-binding protein 52 (not FK506-binding protein12) mediates the neurotrophic action of FK506. J Pharmacol Exp Ther289:1202–1210.

Guo X, Dillman JF, Dawson VL, Dawson TM. 2001a. Neuroimmunophi-lins: novel neuroprotective and neuroregenerative targets. Ann Neurol50:6–16.

Guo X, Dawson VL, Dawson TM. 2001b. Neuroimmunophilin ligandsexert neuroregeneration and neuroprotection in midbrain dopaminergicneurons. Eur J Neurosci 13:1683–1693.

Hamilton GS, Steiner JP. 1998. Immunophilins: beyond immunosuppres-sion. J Med Chem 41:5119–5143.

Harrar Y, Bellini C, Faure JD. 2001. FKBPs: at the crossroads of folding andtransduction. Trends Plant Sci 6:426–431.

Hu BR, Liu CL, Ouyang Y, Blomgren K, Siesjo BK. 2000. Involvementof caspase-3 in cell death after hypoxia-ischemia declines during brainmaturation. J Cereb Blood Flow Metab 20:1294–1300.

Jin K, Graham SH, Mao X, Nagayama T, Simon RP, Greenberg DA. 2001.Fas (CD95) may mediate delayed cell death in hippocampal CA1 sectorafter global cerebral ischemia. J Cereb Blood Flow Metab 21:1411–1421.

Kato H, Oikawa T, Otsuka K, Takahashi A, Itoyama Y. 2000. Postischemicchanges in the immunophilin FKBP12 in the rat brain. Brain Res MolBrain Res 84:58–66.

Khan Z, Ferrari G, Kasper M, Tonge DA, Steiner JP, Hamilton GS,Gordon-Weeks PR. 2002. The non-immunosuppressive immunophilinligand GPI-1046 potently stimulates regeneration axon growth from adult

mouse dorsal root ganglia cultured in matrigel. Neuroscience 114:601–609.

Kitagawa H, Hayashi T, Mitsumoto Y, Koga N, Itoyama Y, Abe K. 1998.Reduction of ischemic brain injury by topical application of glial cellline-derived neurotrophic factor after permanent middle cerebral arteryocclusion in rats. Stroke 29:1417–1422.

Kuwana T, Smith JJ, Muzio M, Dixit V, Newmeyer DD, Kornbluth S.1998. Apoptosis induction by caspase-8 is amplified through the mito-chondrial release of cytochrome c. J Biol Chem 273:16589–16594.

Lyons WE, Steiner JP, Snyder SH, Dawson TM. 1995. Neuronal regen-eration enhances the expression of the immunophilin FKBP-12. J Neu-rosci 15:2985–2994.

Marks AR. 1996. Cellular functions of immunophilins. Physiol Rev 76:631–649.

Matsumura H, Shimizu Y, Ohsawa Y, Kawahara A, Uchiyama Y, NagataS. 2000. Necrotic death pathway in Fas receptor signaling. J Cell Biol151:1247–1255.

Matsushita K, Wu Y, Qiu J, Lang-Lazdunski L, Hirt L, Waeber C, HymanGP, Yuan J, Moskowitz MA. 2000. Fas receptor and neuronal cell deathafter spinal cord ischemia. J Neurosci 20:6879–6887.

Mattson MP, Culmsee C, Yu ZF. 2000. Apoptotic and antiapoptoticmechanism in stroke. Cell Tissue Res 301:173–187.

Moss SJ, Birkestrand B, Fowler SC. 2002. The neuroimmunophilin GPI-1046 partially protects against 3-acetylpyridine toxicity in the rat. Neu-rosci Lett 321:53–56.

Noshita N, Sugawara T, Fujimura M, Morita-Fujimura Y, Chan PH.2001. Manganese superoxide dismutase affects cytochrome c release andcaspase-9 activation after transient focal cerebral ischemia in mice. J CerebBlood Flow Metab 21:557 –567.

Parker EM, Monopoli A, Ongini E, Lozza G, Babij CM. 2000. Rapamy-cin, but not FK506 and GPI-1046, increases neurite outgrowth in PC12cells by inhibiting cell cycle progression. Neuropharmacology 39:1913–1919.

Sauer H, Francis JM, Jiang H, Hamilton GS, Steiner JP. 1999. Systemictreatment with GPI-1046 improves spatial memory and reverse cholin-ergic neuron atrophy in the medial septal nucleus of aged mice. Brain Res842:109–118.

Snyder SH, Lai MM. 1998. Immunophilins in the nervous system. Neuron21:283–294.

Srinivasula SM, Ahmad M, Fernandes-Alnemri T, Litwack G, Alnemri ES.1996. Molecular ordering of the Fas-apoptotic pathway: the Fas/APO-1protease Mch5 is a CrmA-inhibitable protease that activates multipleCed-3/ICE–like cysteine proteases. Proc Natl Acad Sci USA 93:14486–14491.

Steiner JP, Hamilton GS, Ross DT, Valentine HL, Guo H, Connolly MA,Liang S, Ramsey C, Li JH, Huang W, Howorth P, Soni R, Fuller M,Sauer H, Nowotnik AC, Suzdak PD. 1997. Neurotrophic immunophilinligands stimulate structural and functional recovery in neurodegenerativeanimal models. Proc Natl Acad Sci USA 94:2019–2024.

Xu J, Culman J, Blume A, Brecht S, Gohlke P. 2003. Chronic treatmentwith a low dose of lithium protects the brain against ischemic injury byreducing apoptotic death. Stroke 34:1287–1292.

Neuroprotection of GPI-1046 in MCAO 389