Peripheral benzodiazepine receptor ligand PK11195 reduces microglial activation and neuronal death in quinolinic acid-injected rat striatum

www.elsevier.com/locate/ynbdi Neurobiology of Disease 20 (2005) 550 – 561

Peripheral benzodiazepine receptor ligand PK11195 reduces microglial activation and neuronal death in quinolinic acid-injected rat striatum Jae K. Ryu,a Hyun B. Choi,a,b and James G. McLarnona,* a

Department of Pharmacology and Therapeutics, Faculty of Medicine, 2176 Health Sciences Mall, The University of British Columbia, Vancouver, BC, Canada V6T 1Z3 b Division of Neurology, Department of Medicine, Faculty of Medicine, The University of British Columbia, Vancouver, BC, Canada V6T 2B5 Received 7 February 2005; revised 18 March 2005; accepted 8 April 2005 Available online 23 May 2005

The effects of the peripheral benzodiazepine receptor (PBR) ligand, PK11195, were investigated in the rat striatum following the administration of quinolinic acid (QUIN). Intrastriatal QUIN injection caused an increase of PBR expression in the lesioned striatum as demonstrated by immunohistochemical analysis. Double immunofluorescent staining indicated PBR was primarily expressed in ED1-immunoreactive microglia but not in GFAP-immunoreactive astrocytes or NeuNimmunoreactive neurons. PK11195 treatment significantly reduced the level of microglial activation and the expression of pro-inflammatory cytokines and iNOS in QUIN-injected striatum. Oxidative-mediated striatal QUIN damage, characterized by increased expression of markers for lipid peroxidation (4-HNE) and oxidative DNA damage (8-OHdG), was significantly diminished by PK11195 administration. Furthermore, intrastriatal injection of PK11195 with QUIN significantly reduced striatal lesions induced by the excitatory amino acid and diminished QUIN-mediated caspase-3 activation in striatal neurons. These results suggest that inflammatory responses from activated microglia are damaging to striatal neurons and pharmacological targeting of PBR in microglia may be an effective strategy in protecting neurons in neurological disorders such as Huntington’s disease. D 2005 Elsevier Inc. All rights reserved. Keywords: PK11195; Peripheral benzodiazepine receptors; Neuroprotection; Quinolinic acid; Microglia; Inflammation; Oxidative damage

Introduction Excitotoxic processes contribute to the progressive cell death in Huntington’s disease (HD), an inherited neurodegenerative disorder. Studies of brains from HD patients have revealed a relatively selective loss of neurons expressing the glutamate subtype N-

* Corresponding author. Fax: +1 604 822 6012. E-mail address: [email protected] (J.G. McLarnon). Available online on ScienceDirect (www.sciencedirect.com). 0969-9961/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2005.04.010

methyl-d-aspartate (NMDA) receptor (Young et al., 1988). In HD animal models, intrastriatal injection of NMDA receptor agonists such as quinolinic acid (QUIN) leads to characteristic pathological responses resembling those found in HD brain (Beal et al., 1991; Schwarcz et al., 1983). Recent studies have documented the enhanced vulnerability of striatal neurons to excitotoxic injury in cells expressing mutant huntingtin protein (Li et al., 2003; Zeron et al., 2001) and increased level of quinolinate in neostriatum and cortex of early grade HD (Guidetti et al., 2004). Microglia, the resident immune responsive cells of the brain, constitute an important source for the production of excitotoxins. Upon activation with inflammatory stimuli, microglia can release high amounts of QUIN (Espey et al., 1997; Heyes et al., 1996) and glutamate (Piani et al., 1992). Elevated levels of QUIN have been reported following brain damage in vivo with immunoreactivity of the excitotoxin localized to microglia (Lehrmann et al., 2001). Results from immunohistochemical analysis have also provided evidence for the progressive accumulation of reactive microglia in affected regions of HD brain (Sapp et al., 2001). At present, however, the specific roles of microglial responses in the pathology of HD are not well understood. Activated microglia show high expressions of the peripheral benzodiazepine receptor (PBR) located on the outer membrane of mitochondria. PBR have been implicated in a host of cellular functions including regulation of mitochondrial lipid metabolism, apoptosis, cell proliferation, and immune system function (Casellas et al., 2002; Gavish et al., 1999). Importantly, autoradiographic data has established increased PBR density in an excitotoxic rat model of HD (Belloli et al., 2004; Levivier and Przedborski, 1998) and in affected striatum of HD patient brains (Messmer and Reynolds, 1998; Schoemaker et al., 1982). Ligands for the PBR include PK11195 (1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline-carboxamide) and this compound has been shown to possess anti-inflammatory actions in vitro and in vivo. PK11195 inhibited the secretion of pro-inflammatory cytokines (Klegeris et al., 2000) and proliferation of monocytes (Bessler et al.,

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1992) and reduced the production of NO and TNF-a with lipopolysaccharide (LPS) stimulation of cultured rodent microglia (Wilms et al., 2003). In this laboratory, we have documented PK11195 to block the expression and production of TNF-a and COX-2 induced with LPS treatment of human microglia (Choi et al., 2002). In vivo, PBR-specific ligands including PK11195 have reported therapeutic effects in animal models of inflammatory disorders such as rheumatoid arthritis (Waterfield et al., 1999), carrageenan-induced pleurisy (Torres et al., 2000) and pulmonary inflammation (Bribes et al., 2003). These clinical and experimental data raise the possibility that modulation of PBR may have utility to inhibit neuronal degeneration in HD. In this study we have used an excitotoxin rat model of HD, with injection of QUIN into rat striatum, to investigate the effects of PK11195 to block microglial inflammatory responses and to confer neuroprotection. The results suggest a wide-spectrum of efficacy for PK11195 including inhibition of microglial-mediated inflammatory responses, resulting in reduction of pro-inflammatory cytokines and products of lipid peroxidation and oxidative DNA damage, and blockade of QUIN-induced caspase-3 activation and degeneration of striatal neurons.

Materials and methods Animal surgery and drug administration All animal procedures were approved by the University of British Columbia Animal Care Ethics Committee, adhering to guidelines of the Canadian Council on Animal Care. Adult male Sprague – Dawley rats (Charles River Laboratories, St. Constant, Quebec) weighing 250 – 280 g were used in these experiments. The animals were maintained in a temperature and humidity controlled environment under a 12 h light – dark cycle with food and water available ad libitum. The stereotaxic injection of quinolinic acid (QUIN) has been previously described (Ryu et al., 2004). In brief, animals were anesthetized by intraperitoneal (i.p.) injection of a mixture of ketamine hydrochloride (72 mg/kg; Bimeda-MTC, Cambridge, Ontario, Canada) and xylazine hydrochloride (9 mg/ kg; Bayer Inc., Etobicoke, Ontario, Canada) and placed in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA). The animals were unilaterally injected with 60 nmol QUIN (Sigma, St. Louis, MO; dissolved in 0.1 M PBS, pH 7.2) into the striatum at the following coordinate: (AP: +1.0 mm, ML: 3.0 mm, DV: 5.0 mm from bregma, according to Paxinos and Watson, 1986). In a previous work, we have employed 60 nmol of QUIN (Ryu et al., 2003, 2004). We find that this dose (also used by Qin et al., 2001) induces considerable neuropathology and inflammatory reactivity. The injection syringe was left in place for an additional 5 min to avoid backflow of the QUIN. After removing the needle, the skin was sutured and animals allowed to recover and returned to their cages. PK11195 (Sigma) was dissolved in DMSO solution with dilution to a stock concentration of 50 mM in 0.1 M phosphate buffer saline (PBS, pH 7.4). Stock solution was kept in the dark at 4-C. Further dilution with PBS was performed immediately before the injection. PK11195 was stereotaxically injected with QUIN at doses of 0.5 – 10 nmol (1 Al over 5 min) using a 10-Al Hamilton syringe attached to a 26-gauge needle. Displacement of labeled [3H]PK11195 has been used to infer specific binding of PK11195 (nanomolar doses) to the rat PBR (Anholt et al., 1986; Banati et al.,

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2000). Animals injected with only QUIN received the vehicle of PK11195. Intrastriatal injection of PK11195 was used to ensure delivery of the compound into striatum and minimize effects of distribution into other tissue. It can be noted that the doses used are considerably lower than levels shown to cause toxicity in animal studies (Yamada et al., 1999). Previous work has also employed i.p. injection of PK11195 at doses from 0.1 to 25 mg/kg (Torres et al., 2000; Waterfield et al., 1999; Yamada et al., 1999). The caspase-3 inhibitor, Ac-DEVD-CHO (Calbiochem, La Jolla, CA) was dissolved in DMSO and diluted in PBS (The final concentration of DMSO was 0.5%). Ac-DEVD-CHO (at 4 Ag) was administered into striatum with QUIN. Animals injected with only QUIN received the vehicle of Ac-DEVD-CHO. The injected concentration of Ac-DEVD-CHO is the same employed in previous work (Qin et al., 2000). Tissue preparation Animals were sacrificed at 1, 6, 12 h, or 1, 2, or 7 days following QUIN injection. The animals were deeply anesthetized and then transcardially perfused with heparinized cold saline followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (0.1 M PB, pH 7.4). The brains were removed from the skull and postfixed in the same fixative solution overnight and then placed in 30% sucrose in 0.1 M PB for cryoprotection. The brains were then frozen in powdered dry ice and stored at 70-C. Coronal brain sections (40 Am) were cut on a cryostat and stored in cryoprotectant solution. Immunohistochemistry Free-floating sections from different time points were processed for the immunohistochemistry as described previously (Ryu et al., 2004). Briefly, free-floating sections were quenched with 3% hydrogen peroxide in 0.1 M PBS and incubated in blocking solution containing 0.5% bovine serum albumin (BSA), 10% normal goat serum (NGS) and 0.2% Triton X-100 in 0.1 M PBS for 1 h. The sections were then incubated at 4-C for 24 h with the following primary antibodies against peripheral benzodiazepine receptor (PBR; 1:200; Santa Cruz Biotechnology, Santa Cruz, CA), neuronal nuclei (NeuN; 1:1000; Chemicon, Temecula, CA), ED1 (1:500; Serotec, Oxford, UK), complement receptor type 3 (OX-42; 1:500; Serotec), glial fibrillary acidic protein (GFAP; 1:500; Sigma), 4-hydroxynonenal (4-HNE; 1:500; Jaica, Shizuoka, Japan), and 8-hydroxy-2-deoxyguanosine (8-OHdG; 1:500; Jaica). Sections were incubated at RT for 2 h with biotinylated goat anti-mouse or anti-rabbit IgG (1:200; Vector Laboratories, Burlingame, CA). Sections were then incubated for 2 h with the avidin – biotin complex (ABC Elite kit; 1:200; Vector Laboratories) and the reaction was developed with 3,3V-diaminobenzidine (DAB, Sigma) and hydrogen peroxide. Sections were washed in 0.1 M PB, placed on Superfrost/Plus microscope slides (Fisher Scientific; Pittsburgh, PA), dehydrated, and mounted in DPX Mountant (Fluka, Toronto, Canada). Double immunofluorescent staining For double immunofluorescent staining (Ryu et al., 2003), freefloating sections were blocked for 1 h and incubated for 48 h at 4-C with a mixture of two primary antibodies; PBR (1:200; Santa Cruz Biotechnology) in combination with NeuN (1:1000; Chemicon), ED1 (1:500; Serotec) or GFAP (1:1000; Sigma); Cleaved caspase-3

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(1:200; Cell Signaling Technology Inc., Beverly, MA) in combination with NeuN (1:1000; Chemicon). Sections were then incubated with fluorescence-conjugated goat secondary antibodies (Alexa Fluor antibodies; Molecular Probes, Eugene, OR) for 2 h at RT in the dark. Double stained sections were examined under a Zeiss Axioplan 2 fluorescent microscope (Zeiss) equipped with a DVC camera (Diagnostic Instruments). RT-PCR analysis Twenty-four hours after QUIN injection in the absence or presence of PK11195, animals were deeply anesthetized and sacrificed by decapitation. Brains were removed and the striatum was dissected onto a cold metal tissue matrices (Harvard Apparatus, Montreal, Quebec, Canada). The tissue samples were frozen in liquid nitrogen. Total RNA was then extracted from frozen striatal tissue using TRIzol reagent (Life Tech-BRL, Gaithersburg, MD) according to the manufacturer’s protocol. RT-PCR was performed as described previously (Choi et al., 2002). Briefly, reverse transcription was performed using Maloney-murine leukemia virus (M-MLV) reverse transcriptase (Life Tech-BRL). cDNA was amplified by PCR using the PCR primers. Primers with expected product size were as follows: IL-1h, 5V- TGA TGT TCC CAT TAG ACA GC-3V(sense) and 5V-GAG GTG CTG ATG TAC CAG TT3V(antisense) (378 bp); IL-6, 5V-AAA ATC TGC TCT GGT CTT CTG G-3V(sense) and 5V-GGT TTG CCG AGT AGA CCT CA3V(antisense) (300 bp); TNF-a, 5V-TTC TGT CTA CTG AAC TTC GGG GTG ATC GGT CC-3V(sense) and 5V-GTA TGA GAT AGC AAA TCG GCT GAC GGT GTG GG-3V(antisense) (354 bp); iNOS, 5V-CCT GCC CCT TCA ATG GT-3V(sense) and 5V-GGT ATG CCC GAG TTC TTT-3V(antisense) (758 bp); h-actin, 5V-GTG GGG CGC CCC AGG CAC CA-3V(sense) and 5V-GTC CTT AAT GTC ACG CAC GAT TTC-3V(antisense) (526 bp). PCR products were separated by electrophoresis in 1.5% agarose gels containing ethidium bromide and detected under UV light. The intensities of each band were measured by the densitometry using the NIH Image J 1.24 software (National Institutes of Health, Bethesda, MD, USA) as described previously (Franciosi et al., 2005). The band intensities of PCR products were expressed as relative mRNA levels (mRNA values normalized to h-actin).

on sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. After electrophoresis, the separating gel was transfer to polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were blocked in 3% bovine serum albumin in Tris-buffered saline (50 mm Tris base, 150 mm NaCl, 0.5% Triton x-100 (v/v), pH 7.4). After blocking, membranes were then incubated overnight at 4-C with polyclonal anti-rabbit cleaved caspase-3 antibody (Cell Signaling Technology, 1:500). The membranes were then rinsed and incubated with horseradish peroxidase (HRP) conjugated anti-rabbit IgG secondary antibody (Amersham-Pharmacia Biotech, NJ, USA) diluted 1:2000 for 1 h at RT. The blots were developed with an enhanced chemiluminescence kit (ECL, Amersham-Pharmacia Biotech), and signals were captured by Fluor-S MultiImager and quantified using Quantity One software (Bio-Rad, Hercules, CA, USA). Quantitative analysis Several studies, including previous work from this laboratory, have used a time point of 7 days post-QUIN injection for data analysis. In the present study we only used a single application of PK11195 (injected with QUIN) which could lose some efficacy

Western blot analysis Twenty-four hours after injection, protein was extracted from rats treated with QUIN or subjected to QUIN injection with AcDEVD-CHO or PK11195. Striatum was dissected onto a cold metal tissue matrices (Harvard Apparatus) and frozen at 70-C. Frozen striatal tissue was lysed by sonication on ice in homogenization buffer containing 20 mM Tris – HCl, pH 7.4, 2 mM EGTA, 5 mM EDTA, 20 mM 3-(N-morpholino) propane sulfonic acid (MOPS), 30 mM sodium fluoride, 40 mM h-glycerophosphate, 20 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 3 mM benzamidine, 5 AM pepstatin, and 10 AM leupeptin. After sonication, tissue lysate was ultracentrifuged at 100,000g for 30 min at 4-C. The supernatant was collected and stored at 70-C until use. Protein concentration was quantified using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA). Protein samples (50 Ag) were diluted with sample buffer (120 mM Tris – HCl, pH 6.8; 4% SDS (w/v); 20% glycerol (v/v), 10% h-mercaptoethanol, 0.01% bromophenol blue (w/v), denatured by boiling for 5 min, loaded and resolved

Fig. 1. PBR expression in QUIN-injected striatum. (A) Representative photographs of PBR immunoreactivity in lesioned areas of striatal sections. Sections were prepared from nonlesioned control rats (Cont) and QUINinjected rats (1, 6, 12, 24 h and 7 days post-injection). (B) Double immunofluorescent staining of PBR (green) and ED1 (red, left panel) or GFAP (red, right panel) in lesioned striatum taken from rats injected with QUIN (24 h post-injection). Scale bars in A and B are 50 Am.

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after 1 week. The analysis of 1 day post-injection was used for expression of pro-inflammatory cytokines and caspase-3 which are rapidly expressed under inflammatory conditions. We used 2 days post-injection for gliosis (OX-42 and GFAP markers) and neurons (NeuN marker) since previous work (Ryu et al., 2003, 2004) had established considerable changes in these cellular responses at this time point post-QUIN injection. The four immunostained sections (AP: +1.4, +1.2, +1.0, and +0.8) were digitized and analyzed using the image analysis program NIH version 1.57 (Wayne Rasband, NIH) as described previously (Ryu et al., 2004). Neuronal damaged area was assessed in NeuN stained sections. The extent of neuronal damage has been previously evaluated by measuring the NeuNimmunoreactive neuron depleted area (de Almeida et al., 2002). To assess areas of oxidative damage, the same analysis method was employed on 4-HNE (indicative of lipid peroxidation) and 8-OHdG (indicative of oxidative DNA damage) stained sections as descried above. Quantification of damaged neurons in the striatum was performed by counting neurons double stained with NeuN and cleaved caspase-3 antibody under Zeiss Axioplan 2 fluorescent microscope using 400 objective. The mean number of doublelabeled neurons was calculated and expressed as the number of cleaved caspase-3-immunoreactive neurons/mm2. For quantification of gray level of OX-42 (indicative of microglia) and GFAP (indicative of astrocytes), four stained sections were digitized and mean gray level of immunoreactivity was measured and quantified

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using the NIH Image 1.57 program (Acarin et al., 2001). Immunostained sections were digitized using a DVC camera attached to a Zeiss Axioplan 2 microscope. The digitized images were analyzed with Northern Eclipse software. All quantitative analyses were carried out in a blinded manner and four matched sections from each animal were used. Statistical analysis All data are presented as means T SEM. Statistical significance of differences between groups was assessed using one-wayANOVA and Newman – Keuls post hoc multiple comparison test. Significance was set at P < 0.05.

Results Increased PBR expression in QUIN-injected striatum Immunohistochemical procedures were initially used to examine peripheral benzodiazepine receptor (PBR) protein expression after intrastriatal QUIN injection (60 nmol). As shown in Fig. 1A, immunoreactivity for PBR was minimal in nonlesioned brain. Following QUIN-injection, levels of PBR immunoreactivity increased progressively from 1 to 24 h. At 7 days post-QUIN

Fig. 2. Effect of PK11195 on QUIN-induced microglial activation. (A) High (upper panel) and low (lower panel) magnification photographs of OX-42immunostained striatal sections. The treatments shown are for the striatum of nonlesioned control rats, QUIN-injected rats (at 48 h post-injection) in the absence or presence of 5 nmol PK11195 and rats injected with 5 nmol PK11195 alone. (B) Quantification of the optical density of OX-42 immunoreactivity in striatum. Data are mean T SEM (n = 4 animals per group). *P < 0.05 compared with control. #P < 0.05 compared with QUIN-injected rats. Scale bars in A are (upper panel) 1.5 mm and (lower panel) 70 Am.

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injection, some PBR-immunoreactive cells were still apparent in the lesioned striatum (Fig. 1A). The increased PBR immunoreactivity in the QUIN-injected striatum prompted us to examine the specific cell types expressing PBR protein. Double immunofluorescent staining was performed on brain sections from the lesioned striatum at 24 h after QUIN injection. As shown in Fig. 1B, PBR was highly expressed in ED1immunoreactive microglia (left panel) but only weakly present in GFAP-immunoreactive astrocytes (right panel). PBR immunoreactivity was not observed in NeuN-immunoreactive neurons (data not shown). Microglial response in QUIN-injected striatum after PK11195 treatment We next examined whether PK11195 blocks microglial activation induced by QUIN injection. In nonlesioned control brain, OX42-immunoreactive microglia generally displayed small cell bodies with thin processes (Fig. 2A, first panel), a morphology typical of resting cells (Tomas-Camardiel et al., 2004). However, QUIN (48 h post-injection) caused a marked change in cell shape to an enlarged cell body with short and thick processes (Fig. 2A, second panel), a morphology consistent with an activated state (TomasCamardiel et al., 2004). Co-injection of 5 nmol PK11195 with QUIN decreased microglial activation with cells exhibiting a morphology (Fig. 2A, third panel) indicative of a less activated

state compared to that in QUIN injection. In addition to altered shapes of individual microglia, QUIN induced a proliferative response with numbers of cells increased from nonlesioned control (Fig. 2A, first/second panels). PK11195 was highly effective in reducing the numbers of activated microglia (Fig. 2A, third panel). PK11195 injection itself did not induce microglial activation (Fig. 2A, fourth panels). Quantification of overall OX-42 immunoreactivity was performed using measurement of optical density in striatum. As shown in Fig. 2B, QUIN injection significantly increased OX-42 immunoreactivity by 228% relative to nonlesioned control. The corresponding value of immunoreactivity with PK11195 applied with QUIN represented a significant 30% decrease compared with QUIN alone. Injection of PK11195 separately had no effect to alter microglial immunoreactivity compared with nonlesioned control (Fig. 2B). Astroglial response in QUIN-injected striatum after PK11195 treatment To assess the effect of PK11195 on QUIN-induced astrogliosis, sections immunostained with GFAP antibody were analyzed. GFAP-immunoreactive astrocytes (48 h post-QUIN) displayed a morphology consistent with reactivity showing enlarged cell bodies with elongated and thick processes (Fig. 3A, second panel) relative to nonlesioned control (Fig. 3A, first panel). Additionally, number of

Fig. 3. Effect of PK11195 on QUIN-induced astrogliosis. (A) High (upper panel) and low (lower panel) magnification photographs of GFAP-immunostained striatal sections. Treatments shown are for nonlesioned control rats, rats injected with QUIN at 48 h post-injection in the absence or presence of 5 nmol PK11195 and rats injected with PK11195 alone. (B) Quantification of the optical density of GFAP immunoreactivity in striatum. Data are mean T SEM (n = 4 animals per group). *P < 0.05 compared with control. Scale bars in A are (upper panel) 1.5 mm and (lower panel) 70 Am.

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Fig. 4. Effect of PK11195 on QUIN-induced expression of pro-inflammatory cytokines and iNOS mRNA. (A) Representative RT-PCR analysis for IL-1h, IL-6, TNF-a and iNOS mRNA of striatal tissue from nonlesioned control rats and rats injected with QUIN at 24 h in the absence or presence of 5 nmol of PK11195 and rats injected with PK11195 alone. (B) Quantification of relative increases in IL-1h, IL-6, TNF-a and iNOS mRNA induced by QUIN in the absence or presence of PK11195. Data are mean T SEM (n = 4 animals per group). *P < 0.05 compared with control. #P < 0.05 compared with QUIN-injected rats.

astrocytes was markedly increased in striatum with QUIN injection. However, treatment with 5 nmol PK11195 had no evident effect to alter QUIN-induced astrogliosis and the morphology of cells still exhibited a profile of reactivity (Fig. 3A, third panel). Injection of PK11195 alone did not increase astrogliosis (Fig. 3A, fourth panels). Measurement of GFAP immunoreactivity demonstrated that QUIN injection increased astrogliosis by 66% relative to nonlesioned control. PK11195 treatment with QUIN had only a small effect to reduce immunoreactivity compared with QUIN alone which was not statistically significant (Fig. 3B). Weak immuno-

reactivity for GFAP was observed in the PK11195 injected striatum (Fig. 3B). In summary, although intrastriatal QUIN induces microgliosis and astrogliosis, PK11195 was effective as an inhibitor of the microglial, but not astrocyte, response. Effects of PK11195 on pro-inflammatory cytokines and iNOS in QUIN-injected striatum Activated microglia produce a host of inflammatory mediators which are putative neurotoxic factors (McGeer and McGeer, 1995).

Fig. 5. Effect of PK11195 on QUIN-induced lipid peroxidation. (A) 4-HNE immunostaining in the striatum of nonlesioned control rats, QUIN-injected rats at 24 h in the absence or presence of 5 nmol PK11195 and rats injected with 5 nmol PK11195 alone (upper panel). High magnification photographs of 4-HNEimmunostained striatal section (lower panel). (B) Quantification of 4-HNE-immunostained area. Data are mean T SEM (n = 4 animals per group). *P < 0.05 compared with control. #P < 0.05 compared with QUIN-injected rats. Scale bars = 1.5 mm (upper panel) and 70 Am (lower panel).

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We investigated the effect of PK11195 on expression of proinflammatory cytokines (IL-1h, IL-6, TNF-a) and the enzyme inducible nitric oxide synthase (iNOS) after QUIN injection. Representative RT-PCR results revealed little or no expression of IL-1h, IL-6, TNF-a and iNOS mRNA in nonlesioned control (Fig. 4A). However, at a period of 24 h post-QUIN injection, all inflammatory mediators were highly expressed (Fig. 4A). In contrast, animals receiving PK11195 (5 nmol) with QUIN showed marked reductions in expression of IL-1h, IL-6, TNF-a, and iNOS mRNA compared with QUIN alone. PK11195 applied separately had no effect to increase expression of these factors (Fig. 4A). Densitometry analysis of PCR product band intensities is presented in Fig. 4B. Relative to control, intrastriatal QUIN caused significant induction of all factors: IL-1h (by 19-fold), IL-6 (by 14fold), TNF-a (by 54-fold), and iNOS (by 13-fold). Treatment of PK11195 markedly reduced this QUIN-induced expression of IL1h (by 87%), IL-6 (by 65%), TNF-a (by 81%), and iNOS mRNA (by 65%) compared with QUIN alone. Oxidative damage in QUIN-injected striatum after PK11195 treatment

Immunohistochemical analysis showed that 4-HNE-immunoreactivity was not detectable in nonlesioned control (Fig. 5A, first panel). Intrastriatal injection of QUIN caused extensive 4-HNEimmunoreactivity at 24 h post-injection (Fig. 5A, second panel). Co-injection of PK11195 with QUIN led to reduction in the level of 4-HNE (Fig. 5A, third panel). Quantification of 4-HNE-immunostained areas in striatum showed a significant reduction of lipid peroxidation in PK11195-injected animals (Fig. 5B, by 54%) relative to QUIN alone; PK11195 alone had no effect to increase lipid peroxidation (Fig. 5B). QUIN injection into striatum was associated with a marked increase in oxidative DNA damage (24 h post-injection) relative to nonlesioned striatum. Representative 8-OHdG immunohistochemical staining is presented in Fig. 6A (Control, first panel; QUIN, second panel). However, co-injection of PK11195 with QUIN led to a reduction in the 8-OHdG-immunostained area (Fig. 6A, third panel). Quantitative analysis of 8-OHdG immunostaining showed that PK11195 treatment reduced the QUIN-induced 8-OHdG immunoreactive area by 30% compared with QUIN alone (Fig. 6B); PK11195 applied separately had no effect on 8-OHdG immunoreactive area (Fig. 6B). PK11195 increases neuronal survival in QUIN injected striatum

Enhanced levels of oxidative factors have been reported in QUIN-injected striatum (Ryu et al., 2004; Santamaria et al., 2001). To investigate oxidative stress in this model, we examined changes in 4-HNE (indicative of lipid peroxidation) and 8-OHdG (indicative of oxidative DNA damage) in QUINinjected striatum in the absence and presence of PK11195.

An important question addressed in this study considered possible effects of PK11195 to confer neuroprotection in QUINinjected striatum. In these experiments, PK11195 was tested at several concentrations (0.5, 1, 2.5, 5, and 10 nmol and co-injected with QUIN). Overall, striatal damage was examined at 48 h post-

Fig. 6. Effect of PK11195 on QUIN-induced oxidative DNA damage. (A) 8-OHdG immunostaining in the striatum of nonlesioned control rats, QUIN-injected rats at 24 h in the absence or presence of 5 nmol PK11195 and rats injected with 5 nmol PK11195 alone (upper panel). High magnification photographs of 8-OHdG-immunostained striatal section (lower panel). (B) Quantification of 8-OHdG-immunostained area. Data are mean T SEM (n = 4 animals per group). *P < 0.05 compared with control. #P < 0.05 compared with QUIN-injected rats. Scale bars = 1.5 mm (upper panel), 70 Am (lower panel).

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injection using immunohistochemistry for NeuN (indicative of neurons). As shown in Fig. 7A (upper first/second panels), QUINinjected striatum exhibited an extensive loss of NeuN-immunoreactive area relative to nonlesioned control. However, co-administration of 5 nmol PK11195 with QUIN significantly increased the NeuN-immunoreactive area in the striatum (Fig. 7A, upper third panel). High magnification of NeuN-stained striatal sections revealed that the density of NeuN-immunoreactive neurons was significantly diminished by QUIN injection (Fig 7A, lower first/ second panels). Application of PK11195 to QUIN-injected striatum was highly effective in increasing the number of NeuN-immunoreactive neurons (Fig. 7A, lower third panel). The depletion of the NeuN-immunoreactive area in striatum, in the absence and presence of PK11195, was measured with results presented in Fig. 7B. At concentrations of 2.5 and 5 nmol, PK11195 showed significant reductions in the depletion of NeuN-immunoreactive areas (by 25% and 49%, respectively) relative to QUIN alone. Lower concentrations of PK11195 (at 0.5 and 1 nmol) produced small decreases, which were not statistically significant, compared with QUIN injection alone. A high concentration of 10 nmol PK11195 was ineffective in blocking QUIN-mediated striatal neuronal damage (data not shown). Thus, PK11195 shows a

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window of significant striatal neuroprotection when injected at concentrations between 2.5 and 5 nmol. To examine if PK11195-induced neuroprotection was correlated with blockade of microglial activation, several experiments were done using a low concentration of PK11195 (at 0.5 nmol). At this dose, PK11195 was ineffective as a neuroprotectant against QUIN toxicity (Fig. 7B). Quantification of OX-42 immunoreactivity was performed as described in Fig. 2 and showed that at 0.5 nmol PK11195 had no effect to inhibit microglial reactivity (Fig. 7C). PK11195 decreases caspase-3 activation in neurons Enhanced caspase-3 activity is considered a factor associated with neuronal cell damage (Du et al., 1997). Double immunofluorescent staining with NeuN (indicative of neurons) and cleaved caspase-3 antibody (indicative of activated form of caspase-3) were performed to investigate whether PK11195-mediated neuroprotection was correlated to the inhibition of caspase-3 activation. Representative photographs of double stained sections, at 24 h post-QUIN injection, are presented in Fig. 8A. In the nonlesioned striatum, double staining of NeuN and cleaved caspase-3 exhibited morphological characteristics of normal viable neurons without

Fig. 7. Effect of PK11195 on QUIN-induced neuronal loss. (A) NeuN immunostaining in the striatum of nonlesioned control rats, QUIN injected rats at 48 h in the absence or presence of 5 nmol PK11195 and rats injected with 5 nmol PK11195 alone (upper panel). High magnification photographs of NeuNimmunostained striatal section (lower panel). (B) Quantification of loss of NeuN-immunoreactive area. Data are mean T SEM (n = 4 animals per group). (C) Quantification of the optical density of OX-42 immunoreactivity in striatum. Little change in OX-42 immunoreactivity was found in 0.5 nmol PK11195injected animals. *P < 0.05 compared with control. #P < 0.05 compared with QUIN-injected rats. Scale bars = 1.5 mm (upper panel), 30 Am (lower panel).

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Fig. 8. Effect of PK11195 on QUIN-induced caspase-3 expression in neurons. (A) Double immunofluorescent staining of NeuN (red) and cleaved caspase-3 (green) in the striatum of nonlesioned control rats, QUIN injected rats at 24 h in the absence or presence of 5 nmol PK11195 and rats injected with 5 nmol PK11195 alone. (B) Quantification of the number of NeuN-immunoreactive neurons expressing cleaved caspase-3. Data are mean T SEM (n = 4 animals per group). *P < 0.05 compared with control. #P < 0.05 compared with QUIN-injected rats. Scale bar = 20 Am.

any expression of the activated form of caspase-3 (Fig. 8A, first panel). Following QUIN injection (Fig. 8A, second panel), NeuN immunohistochemistry demonstrated smaller neuronal cell bodies and non-spherical shapes compared to nonlesioned control. Additionally, excitotoxin injection caused a relatively high level of cleaved caspase-3 immunoreactivity in neurons which was markedly reduced if PK11195 was co-injected with QUIN (Fig. 8A, third panel). Double staining for NeuN and cleaved caspase-3 showed that PK11195 injection itself did not induce cleaved caspase-3 immunoreactivity in neurons (Fig. 8A, fourth panel). The results of quantitative analysis for cleaved caspase-3 expression in neurons are presented in Fig. 8B. In QUIN-injected striatum, the number of cleaved caspase-3-immunoreactive neurons was increased by 254-fold compared with nonlesioned striatum. PK11195 treatment was highly effective in decreasing the number of cleaved caspase-3 expressing neurons by 31% relative to QUIN alone. PK11195 injection itself had no significant effect on numbers of cleaved caspase-3-immunoreactive neurons (Fig. 8B). We further examined, using Western blot analysis, effects of PK11195 on levels of the active form of caspase-3 protein in the QUIN-injected striatum. At 24 h post-QUIN injection, increase in

cleaved caspase-3 protein was observed in the lesioned striatum (Fig. 9A). This QUIN-induced caspase-3 activation was confirmed by stereotaxic injection of the specific caspase-3 inhibitor AcDEVD-CHO; QUIN-induced cleaved caspase-3 protein induction was significantly attenuated by Ac-DEVD-CHO treatment (Fig. 9A). Increased cleaved caspase-3 protein level was also markedly inhibited by PK11195 treatment at 24 h after QUIN injection (Fig. 9A). Densitometry analysis showed that QUIN-induced expression of cleaved caspase-3 protein was inhibited by 56% and 31% in AcDEVD-CHO- and PK11195-treated animals (Fig. 9B). We also examined whether inhibition of caspase-3 activation could block QUIN-induced neuronal damage in striatum. As shown in Fig. 9C, NeuN immunohistochemistry revealed that depleted NeuNimmunoreactive area at 48 h after QUIN injection was markedly decreased by Ac-DEVD-CHO injection (by 37%). Ac-DEVD-CHO alone had no effect on NeuN-immunoreactive area (Fig. 9C).

Discussion The results presented in this study are noteworthy in demonstrating a wide-spectrum of anti-inflammatory actions of PK11195

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Fig. 9. Activation of caspase-3 in QUIN-injected striatum. (A) Representative Western blot analysis for cleaved caspase-3 protein of striatal tissues taken from nonlesioned control rats and rats treated with QUIN or subjected to QUIN injection with Ac-DEVD-CHO or PK11195 at 24 h post-injection. (B) Quantitative analysis of relative increase in cleaved caspase-3 protein. (C) Quantification of the loss of NeuN-immunoreactive area in striatum. Data are mean T SEM (n = 4 animals per group). *P < 0.05 compared with control. #P < 0.05 compared with QUIN-injected rats.

which cumulatively confer neuroprotection against QUIN-induced excitotoxicity. The effects of PK11195 are manifest on a number of factors including inhibition of pro-inflammatory cytokines and iNOS expression and reduction in the levels of indicators for lipid peroxidation and oxidative DNA damage. Overall, the results suggest that actions of PK11195 to inhibit activation of microglia could account for the efficacy of this agent as a neuroprotectant. A novel finding in this work was the up-regulation of PBR in an excitotoxic animal model of Huntington’s disease (HD). Expression of PBR was associated with ED1-immunoreactive cells indicative of resident microglia or infiltrating macrophages; staining for PBR was low in astrocytes and not present in neurons. Although extensive microgliosis and astrogliosis were evident following intrastriatal QUIN injection, PK11195 was effective as an inhibitor of microglial, but not astrocyte, activation. Results from study of other animal models including experimental autoimmune encephalomyelitis (EAE) (Vowinckel et al., 1997), trimethyltin toxicity (Kuhlmann and Guilarte, 2000) and cuprizone-induced demyelin-

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ation (Chen et al., 2004) have demonstrated increased levels of PBR during progression of pathology. Increased expression of PBR in EAE was associated with microglia (Banati et al., 2000; Vowinckel et al., 1997) and in trimethyltin poisoning elevated PBR was found in both microglia and astrocytes (Kuhlmann and Guilarte, 2000). In the latter study, PBR expression for astrocytes was evident for longer times (in excess of 2 weeks) after the brain insult induced by trimethyltin toxicity. Thus, a lack of substantive expression of PBR in astrocytes in QUIN-injected striatum could reflect an analysis at an early time point of 24 h after injection of the excitotoxin. The overall results of this study suggest PK11195 actions on microglia are largely responsible for the decrease in inflammatory mediators and preventing damage to striatal neurons. However, the mechanisms by which PK11195 binding to PBR in microglia inhibits cellular expression of pro-inflammatory cytokines and the enzyme iNOS are not known. Evidently, the PBR on outer mitochondrial membrane is linked to intracellular signal transduction pathways which modulate microglial inflammatory responses. Since PK11195 was effective in blocking the expression of a diversity of QUIN-induced factors, actions on more than one signaling pathway may be involved. A recent review has detailed PK11195 actions on activated microglia (Banati, 2002). At present, it is not known if activation of microglia results from direct stimulatory actions of QUIN (Tikka et al., 2001; Tikka and Koistinaho, 2001; Noda et al., 2000) or alternatively indirectly from excitotoxicity of QUIN on striatal neurons (Lehrmann et al., 2001; Topper et al., 1993). In the latter case, excitotoxic damage to neurons could release signaling factors such as ATP to signal chemotactic responses and subsequent activation of microglia (Inoue, 2002). Excitotoxin injection into brain causes increased levels of proinflammatory cytokines (Yu et al., 2002) and enhanced reactive species such as NO and oxidative stress (Bal-Price and Brown, 2001; Stone et al., 2000). QUIN-injected striatum showed considerable increase in oxidative damage markers (4-HNE and 8-OHdG) relative to nonlesioned control. Treatment with PK11195 was highly effective in blocking the levels of both agents (Figs. 5 and 6). A previous study using QUIN-injected striatum has also demonstrated that inhibition of oxidative stress using the antioxidant pyruvate or the iNOS inhibitor aminoguanidine was neuroprotective (Ryu et al., 2004). Interestingly, oxidative stress was associated with glial responses since pyruvate blocked iNOS and nitrotyrosine in both microglia and astrocytes. The present results suggest that suppression of microglial activation, mediated by PK11195 binding to PBR sites in activated cells, as a mechanism in reducing oxidative damage. At present, it is not known if the elevated levels of 4-HNE and 8-OHdG with QUIN injection are a direct measure of oxidative damage or instead reflect neuronal injury due to other mechanisms. An interesting finding in this work was that PK11195 treatment was effective in reducing neuronal caspase-3 activation (Fig. 8). Application of Ac-DEVD-CHO, a selective caspase-3 inhibitor, also decreased QUIN-induced striatal neuronal damage (Fig. 9). These results would suggest activation of caspase-3 in neurons is a contributing factor in QUIN-induced neurotoxicity. Results from previous work have suggested involvement of caspase-3 in the pathogenesis of neurodegeneration in brain damage including excitotoxic injury (Qin et al., 2000; Tenneti and Lipton, 2000; Du et al., 1997). However, in QUIN-injected striatum, double immunofluorescent labeling analysis showed an absence of PBR in neurons (data not shown). Thus, under our experimental

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conditions, the decrease in neuronal cleaved caspase-3 immunoreactivity (Fig. 8) is unlikely induced by direct actions of PK11195 on neurons. Instead, the finding of PBR localization to microglia (Fig. 1B) lead us to speculate that PK11195 block of microglial activation could reduce neuronal damage by decreasing cleaved caspase-3 levels in neurons. The QUIN model of HD model dose not account for several factors found in the pathogenesis of HD including genetic defects, nuclear inclusions, and a slow disease progression (Blum et al., 2003). However, the acute excitotoxic model does demonstrate striatal pathology resembling that found in the disease. Excitotoxicity and inflammatory responses induced by QUIN injection may mimic the cellular responses during certain stages in the progression of HD. This possibility is supported by recent studies with HD postmortem brains revealing enhanced levels of QUIN (Guidetti et al., 2004), complement (Singhrao et al., 1999) and reactive microglia in affected regions (Sapp et al., 2001). In this regard, activated microglia are sources of excitatory amino acids including QUIN which could cause substantive neurodegeneration under pathological conditions (Heyes et al., 2001; Sinz et al., 1998). Overall, our results are consistent with microglial activation and inflammatory responses contributing to neuronal damage in QUINinjected striatum. Elevated expression of PBR in activated microglia may play contributory roles in the production of a milieu of inflammatory mediators. Pharmacological modulation of PBR in activated microglia, by ligands such as PK11195, could serve as a novel neuroprotective strategy, aimed at reducing CNS inflammation in neurological disorders including HD.

Acknowledgments This work was supported by a grant from Alzheimer’s Association USA (to JGM) and a Michael Smith Memorial Fellowship (to JKR).

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