Fluoxetine prevents LPS-induced degeneration of nigral dopaminergic neurons by inhibiting microglia-mediated oxidative stress

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Fluoxetine prevents LPS-induced degeneration of nigral dopaminergic neurons by inhibiting microglia-mediated oxidative stress Eun S. Chung a,b,c,1 , Young C. Chung a,b,c,d,1 , Eugene Bok a,b,c,d , Hyung H. Baik a,b , Eun S. Park a,b,c , Ju-Young Park e , Sung-Hwa Yoon e , Byung K. Jin a,b,c,⁎ a

Department of Biochemistry and Molecular Biology, School of Medicine Kyung Hee University, Seoul 130-701, Republic of Korea Neurodegeneration Control Research Center, School of Medicine Kyung Hee University, Seoul 130-701, Republic of Korea c Age-related & Brain Diseases Research Center, School of Medicine Kyung Hee University, Seoul 130-701, Republic of Korea d Neuroscience Graduate Program, Division of Cell Transformation and Restoration, School of Medicine, Ajou University, Suwon 443-479, Republic of Korea e Department of Molecular Science and Technology, Ajou University, Suwon 443-479, Republic of Korea b

A R T I C LE I N FO

AB S T R A C T

Article history:

Lipopolysaccharide (LPS)-induced microglial activation causes degeneration of nigral

Accepted 14 September 2010

dopaminergic (DA) neurons. Here, we examined whether fluoxetine prevents LPS-induced

Available online 18 September 2010

degeneration of DA in the rat substantia nigra (SN) in vivo. Seven days after LPS injection into the SN, immunostaining for tyrosine hydroxylase (TH) revealed a significant loss of

Keywords:

nigral DA neurons. Parallel activation of microglia (visualized by OX-42 and ED1

Fluoxetine

immunohistochemistry), production of reactive oxygen species (ROS) (assessed by

Parkinson's disease

hydroethidine histochemistry), and degeneration of nigral DA neurons were also

Microglial activation

observed in the SN. Western blot analyses and double-label immunohistochemistry

ROS

showed an increase in the expression of inducible nitric oxide synthase (iNOS) within

iNOS

activated microglia. LPS also induced translocation of p67phox, the cytosolic component of

Oxidative stress

NADPH oxidase, to the membrane of SN microglia, indicating activation of NADPH oxidase. The LPS-induced loss of nigral DA neurons was partially inhibited by fluoxetine, and the observed neuroprotective effects were associated with fluoxetine-mediated suppression of microglial NADPH oxidase activation and iNOS upregulation, and decreased ROS generation and oxidative stress. These results suggest that fluoxetine and analogs thereof may be beneficial for the treatment of neurodegenerative diseases, such as PD, that are associated with microglia-derived oxidative damage. © 2010 Elsevier B.V. All rights reserved.

⁎ Corresponding author. Department of Biochemistry and Molecular Biology, Neurodegeneration Control Research Center, Age-related & Brain Diseases Research Center, School of Medicine Kyung Hee University, Seoul 130-701, Republic of Korea. Fax: + 82 2 969 4570. E-mail address: [email protected] (B.K. Jin). 1 These authors contributed equally to this work. 0006-8993/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2010.09.049

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1.

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Introduction

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by a dramatic loss of nigral dopaminergic (DA) neurons in the substantia nigra (SN). Although the cause of PD and the mechanisms that mediate disease development remain elusive, accumulating clinical and experimental evidence suggests that microglial activation plays a critical role in the pathogenesis of PD through production of neurotoxic and inflammatory mediators (Hirsch et al., 1998; Whitton, 2007). These activated microglia-generated neurotoxins include NADPH oxidase-/iNOS-derived reactive oxygen species (ROS) that, in turn, trigger oxidative stress (Liberatore et al., 1999; Wu et al., 2003). In this context, several indices of oxidative stress (Dawson and Dawson, 2003; Savitt et al., 2006) and activated microglia (Block et al., 2007) are observed in the substantia nigra (SN) of PD patients. In 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine (6OHDA) models of PD, microglial NADPH oxidase-/iNOSderived ROS produce oxidative stress and lead to eventual DA neuronal death in vivo (Liberatore et al., 1999; Singh et al., 2005; Wu et al., 2003). Lipopolysaccharide (LPS)-induced microglial activation causes degeneration of mesencephalic DA neurons both in vivo and in vitro (Arimoto and Bing, 2003; Gao et al., 2003b). In the LPS-induced inflammation model, microglial NADPH oxidase and/or iNOS have been reported to be centrally involved in triggering oxidative stress and mediating neurotoxicity (Arimoto and Bing, 2003; Gao et al., 2003c). Fluoxetine, a selective serotonin reuptake inhibitor, is most commonly prescribed as an antidepressant, but has also been found to exert anti-inflammatory and pain-relieving effects in peripheral systems (Abdel-Salam et al., 2004; Bianchi et al., 1995). Recently, fluoxetine was shown to attenuate postischemic brain damage through suppression of microglia activation (Lim et al., 2009). Additionally, paroxetine, another antidepressant, rescues nigrostriatal dopaminergic neurons from MPTP neurotoxicity by inhibiting brain inflammation and oxidative stress in vivo (Chung et al., 2010). Apart from anti-inflammatory properties of fluoxetine, it has been shown that fluoxetine increased the generation of neural precursor cells in 6-hydroxydopamine (6-OHDA) lesioned rat (Suzuki et al., 2010). However, little is known about the effects of fluoxetine in the central nervous system, especially in mesencephalic DA neurons in the context of PD. Thus, the present study examined whether fluoxetine could protect nigral DA neurons from LPS neurotoxicity by inhibiting microglial activation and reducing subsequent oxidative stress.

2.

Results

2.1.

Neuroprotective effect of fluoxetine in LPS-treated SN

Phosphate-buffered saline (PBS, 3 μl) or LPS (5 mg/3 μl) was unilaterally injected into the SN of the rat brain. Seven days after LPS injection, brains were removed, sectioned, and processed for TH immunostaining to specifically detect DA

neurons (Figs. 1a,b,d,e) and Nissl staining (Figs. 1c,f). A considerable loss of TH-positive cells (Figs. 1d,e) and Nisslstained cells (Fig. 1f) was observed in the LPS-injected SN compared with PBS-injected controls (Figs. 1a–c). Highly magnified microphotographs revealed that LPS induced a

Fig. 1 – Fluoxetine protects DA neuronal death against LPS-induced neurotoxicity. PBS or LPS (5 μg) was unilaterally injected into the SN in the absence or presence of fluoxetine (5 or 10 mg/kg, i.p.). Animals were sacrificed 7 days after LPS injection, brains section was selected and processed for immunostaining with TH antibody (a,b,d,e,g,h) and Nissl staining (c,f,i). (a–c) PBS. (d–f) LPS. (g–i) LPS + fluoxetine (10 mg/kg, i.p.). The lower case letters (b,e,h) indicate higher magnifications of a,d,g, respectively. Scale bar, 400 μm in a,d, g; 50 μm in b,e,h and c,f,i. (j) Number of TH-ip neurons in the LPS-injected SN in the absence or presence of fluoxetine. Six to eight animals were used for each experimental group. The results represent means ± SEM. *p < 0.001 versus PBS-injected SN; #p < 0.05, ##p < 0.001 versus LPS-injected SN. SNpc, substantia nigra pars compacta; VTA, ventral tegmental area.

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remarkable loss of Nissl substances with gliosis (Fig. 1f), and altered the morphology of TH-positive neurons; unlike the large and healthy DA neurons with long and branched neuritic processes observed in PBS-injected SN samples (Fig. 1b), THpositive neurons in the LPS-treated SN were characterized by shrunken neuronal cell bodies (Fig. 1e). To investigate the potential of fluoxetine to prevent LPSinduced neurotoxicity of nigral neurons, we administered fluoxetine (5 or 10 mg/kg, i.p.) twice daily for 9 days starting 2 days before intranigral LPS injection. Fluoxetine significantly attenuated the LPS-induced loss of TH-positive DA neurons in the SN, and preserved a more normal neuronal morphology, as evidenced by relatively healthy cell bodies with branched processes (Figs. 1g–h). In addition, Nissl staining showed that fluoxetine treatment effectively reduced LPS-induced neuronal death in the SN (Fig. 1i). Quantification of TH-positive neurons revealed that fluoxetine exerted a dose-dependent neuroprotective effect, increasing the number of TH-positive neurons (expressed as a percentage of those in the ipsilateral SN) by 12% at 5 mg/ kg (p < 0.05) and by 49% at 10 mg/kg (p < 0.001) compared with the LPS-injected SN (Fig. 1j). Animals that received saline (in the absence of LPS or fluoxetine) or fluoxetine alone (5 or 10 mg/kg) showed no remarkable reduction of DA neurons in the SN (data not shown).

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2.2. LPS-induced microglial activation and oxidant production are inhibited by fluoxetine Several reports, including those from our laboratory, have indicated that LPS activates rat microglia and contributes to neuronal death in vivo (Park et al., 2007; Qin et al., 2007). Thus, we next examined the effect of fluoxetine on LPS-induced microglial activation in the SN. The SN sections adjacent to those used for TH immunostaining in Fig. 1 were processed for immunohistochemical staining using antibodies against OX42 (Figs. 2a,e,i) or ED1 (Figs. 2c,g,k) to detect activated microglia, as described previously (Choi et al., 2003). In the PBS-injected SN, the majority of OX-42-positive microglia exhibited a resting morphology, characterized by small cell bodies with thin, long or ramified processes (Figs. 2a,b). Activated microglia with enhanced OX-42 staining intensity and activated morphology—larger cell bodies with short, thick (or absent) processes—were observed in the LPS-injected SN (Figs. 2e,f). There were also numerous ED1-positive cells in LPS-injected SN tissues (Figs. 2g,h) representing phagocytic microglia, specifically, those that accumulated intracellular lipid vacuoles (Choi et al., 2003). In contrast, ED1-positive cells were restricted to the needle tract in the PBS-injected SN (Figs. 2c,d). Pre-treatment with fluoxetine dramatically decreased the number of activated (OX-42-positive) microglia with an

Fig. 2 – Fluoxetine inhibits LPS-induced microglial activation in the SN. a–l, Sections (a–d, PBS; e–h, LPS; i–l, LPS + fluoxetine) adjacent to those used in Fig. 1 were immunostained with OX-42 (a,b,e,f,i,j) or ED1 (c,d,g,h,k,l) antibodies for microglia. The data are representative of six to eight animals used for each experimental group. b,d,f,h,j,l, Highly magnified areas of OX-42 (a,e,i) and ED1 (c,g,k) immunoreactivity indicated by arrows, respectively. Arrowheads indicate needle tracts. (a, c) PBS. (e, g) LPS. (i, k) LPS + fluoxetine. Scale bar, 400 μm. Dotted lines indicate the SNpc.

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amoeboid shape (Figs. 2i,j) and phagocytic (ED1-positive) activity (Figs. 2k,l). Fluoxetine alone had no effect on microglial activation (data not shown). Recent studies have suggested that activated microglia produce O−2 and O−2-derived oxidants (Block et al., 2007; Koutsilieri et al., 2002). These microglial-derived ROS are thought to mediate the loss of nigral DA neurons in vivo and in vitro (Choi et al., 2005; Gao et al., 2003c; Wu et al., 2003). Thus, we investigated whether fluoxetine enhanced DA neuronal survival by inhibiting LPS-induced ROS production. The fluorescent products of oxidized hydroethidine (i.e., ethidium accumulation) were significantly increased at 48 h in the LPSinjected SN (Fig. 3b) compared with PBS-injected controls

Fig. 3 – Fluoxetine inhibits LPS-induced O−2 and O−2-derived oxidant production and protein oxidation in the SN. Animals received hydroethidine (500 ml PBS containing 2 mg/ml through tail vein) 48 h after intranigral injection of LPS in the absence or presence of fluoxetine. After 45 min, brains were harvested and sections of SN were prepared for hydroethidine histochemistry to detect extracellular superoxide. Confocal microscope observation indicates ethidium fluorescence (red) in the SN treated with (a) PBS, (b) LPS, (c) LPS + fluoxetine. Nuclei were counterstained with Hoechst 33258 (blue). Dotted lines indicate the SNpc, where DA neurons were degenerating after LPS injection. These data are representative of five to six animals per group. Scale bar, 500 μm in a–c. (d) Fluoxetine reduces LPS-induced protein oxidation in the SN. Animals were decapitated 48 h after intranigral injection of LPS in the absence or presence of fluoxetine. Tissue lysates from the ipsilateral SN were analyzed by Western blotting for protein carbonyls as markers of oxidatively modified proteins. (e) Bars represent the means ± SEM of six to seven samples. P, PBS; L, LPS; L + F, LPS + fluoxetine. *p < 0.001 versus PBS-injected SN; #p < 0.001 versus LPS-injected SN.

(Fig. 3a). This LPS-induced oxidant production was dramatically decreased by fluoxetine (Fig. 3c). We also assessed the level of protein carbonyls, a marker of protein oxidation, by densitometric analysis of Western blots. The protein carbonylation level was significantly increased in the SN at 48 h after intranigral injection of LPS compared with PBS-injected controls (Figs. 3d,e). Pre-treatment with fluoxetine significantly reduced the levels of LPS-induced oxidative protein damage in the SN (Figs. 3d,e), but had no effect alone (data not shown).

2.3. Effects of fluoxetine on LPS-induced activation of microglial NADPH oxidase and iNOS NADPH oxidase, the enzyme responsible for oxidant production, is composed of the cytosolic components such as p67phox and the membrane components (Cross and Segal, 2004). Activation of this enzyme, which requires translocation of its cytosolic subunits to plasma membrane, produces ROS in activated microglia and ultimately contributes to DA neuronal death (Gao et al., 2003b,c). Thus, we investigated whether fluoxetine modulated the activity of NADPH oxidase in the LPS-injected SN. Western blot analyses showed that the levels of the cytosolic subunit p67phox were significantly increased in membrane fractions 24 h after LPS injection compared with controls (Fig. 4a), indicating translocation and activation of NADPH oxidase. Moreover, the p67phox-positive cells observed 24 h after LPS injection corresponded to OX-42-positive microglia (Fig. 4c). When fluoxetine was administered before LPS injection, translocation of p67phox from the cytosol to the membrane in the SN was decreased by 58% (p < 0.05; Fig. 4b). Fluoxetine alone had no effects (data not shown). iNOS is upregulated in glial cells in the SN of PD patients (Hunot et al., 1996), and has been implicated in DA neuronal cell death in the SN (Choi et al., 2003; Liberatore et al., 1999). Thus, we examined whether fluoxetine modulated DA neuronal survival by affecting LPS-induced expression of iNOS in the SN. Western blot analyses showed that LPS enhanced the expression of iNOS protein 24 h after injection in the SN (Fig. 4d). Administration of fluoxetine prior to LPS injection reduced iNOS protein levels by 70% (P < 0.05; Fig. 4e). Additionally, double-label immunostaining demonstrated that iNOS immunoreactivity was localized within OX-42-positive microglia (Fig. 4f). Fluoxetine alone had no effects on the level of iNOS expression (data not shown).

3.

Discussion

The present study is the first demonstration that the neuroprotective effects of fluoxetine in the LPS-treated SN are associated with the ability of fluoxetine to suppress microglial NADPH oxidase and iNOS, and thereby decrease ROS generation and oxidative stress. Increasing evidence suggests that activated microglia induce or exacerbate neurotoxicity through the generation of oxidative stressors. DA neurons in the SN show distinctive features, such as depletion of glutathione (Bharath et al., 2002; Sian et al., 1994) or accumulation of total iron (Bharath et al., 2002; Dexter et al., 1989), that make the neurons particularly

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Fig. 4 – (a–c) Fluoxetine inhibits activation of microglial NADPH oxidase. (a) Translocation of cytosolic subunit (p67phox) from the cytosol to the plasma membrane 24 h after intranigral injection of LPS, indicating activation of NADPH oxidase in the SN. Note that this translocation was inhibited by fluoxetine. Tissue lysates from the ipsilateral SN were prepared 24 h after intranigral injection of PBS or LPS in the absence or presence of fluoxetine, fractionated, and analyzed by immunoblot analysis with p67phox antibody. The membrane protein calnexin was used to exhibit fractionation efficiency and normalize the data. (b) Bars represent the means ± SEM of six to seven samples. *p < 0.05 versus PBS-injected SN; #p< 0.05 versus LPS-injected SN. C, Control; P, PBS; L, LPS; L + F, LPS + fluoxetine. (c) Colocalization of p67phox immunoreactivity within the activated microglia in the SN. The sections of rat SN were prepared 24 h after intranigral injection of LPS and then immunostained simultaneously with p67phox (red) and OX-42 (green) as a marker of microglia. Images were captured from the same area and merged. Scale bar, 25 mm. (d–f) Fluoxetine reduces LPS-induced iNOS expression in the SN. (d) Tissue lysates from the ipsilateral SN were prepared 24 h after intranigral injection of PBS or LPS in the absence or presence of fluoxetine. Protein samples were analyzed by immunoblot with iNOS antibody. Note that fluoxetine attenuated LPS-induced iNOS expression. (e) Bars represent the means ± SEM of six to seven samples. *p< 0.05 versus PBS-injected SN; #p < 0.05 versus LPS-injected SN. f, Colocalization of iNOS immunoreactivity within activated microglia in the SN. The sections of rat SN adjacent to those used in panel c were immunostained simultaneously with iNOS (red) and OX-42 (green) as a marker of microglia. Images were captured from the same area and merged. Scale bar, 25 mm.

vulnerable to oxidative stress. Furthermore, the number of microglia is higher in the SN than in other brain regions (Kim et al., 2000). These microglia are quickly activated by various insults (Kreutzberg, 1996), leading to the production of several potentially neurotoxic molecules, including ROS. ROS, such as O−2 and O−2-derived oxidants, can cross cell membranes and induce neuronal death by causing oxidative damage to cellular components, such as proteins (Cadet and Brannock, 1998). The potential clinical significance of this process is highlighted by an earlier postmortem study of PD patients, which showed evidence of oxidative stress, including oxidative modifications to proteins (Floor and Wetzel, 1998). Importantly, these ROS can be generated by microglial-derived NADPH oxidase

and have been shown to cause oxidative stress in vitro and in an in vivo model of PD (Gao et al., 2003a; Wu et al., 2003). In the LPSinduced inflammation model, ROS derived from NADPH oxidase contribute to DA neuronal death in vitro (Gao et al., 2003b) and in vivo (Hernandez-Romero et al., 2008). The results of the present study show that LPS induced translocation of the cytosolic NADPH oxidase subunit p67phox to the plasma membrane, resulting in enhanced extracellular ROS and protein damage in the SN. Treatment with fluoxetine not only inhibited microglial NADPH oxidase activation, but also mitigated ROS production and protein oxidation. Collectively, these results suggest that LPS-induced activation of NADPH oxidase and oxidative stress were prevented by fluoxetine, resulting in neuroprotection.

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In addition to O−2 and O−2-derived oxidants, NO generated by iNOS is also thought to contribute to the oxidative stress associated with the neurodegeneration observed in PD (Hunot et al., 1996; Liberatore et al., 1999). The neurotoxic effects of NO are usually attributed to NO reaction with O−2 to form peroxynitrite, which can cause DA neuronal injuries in 6OHDA and MPTP models of PD (Liberatore et al., 1999; Singh et al., 2005). The present data show that LPS upregulated iNOS expression within activated microglia in the SN, an increase that was inhibited by treatment with fluoxetine. These data support the hypothesis that the observed neuroprotective effects of fluoxetine are associated with the ability of the drug to inhibit microglial iNOS-derived oxidative stress on DA neurons. Together, the present findings demonstrate that fluoxetine may inhibit NADPH oxidase-/iNOS-derived ROS production by activated microglia, preventing oxidative damage to neurons and leading to increased neuronal survival. These results suggest that fluoxetine and analogs thereof hold promise as novel therapeutic agents for neurodegenerative diseases, such as PD, which are associated with microglia-derived oxidative damage.

4.

Experimental procedures

4.1.

Materials

Materials were purchased from the following companies: antityrosine hydroxylase (TH; Pel-Freez, Rogers, AR, USA), mouse anti-OX-42 (specific for complement receptor type 3 (CR3); Serotec, Oxford, UK), mouse anti-ED1 (specific for glycosylated lysosomal antigen; Serotec), monoclonal anti-p67phox (BD Transduction), fluorescein-conjugated lycopersicon esculentum (tomato) lectin (TL; Vector Laboratories, Burlingame, CA, USA), polyclonal anti-iNOS (upstate Biotechnology), hydroethidine (Molecular Probes, Eugene, OR), biotinylated secondary antibody (1:200; KPL, Maryland, USA), an avidin–biotin complex kit (Vectastain ABC Kit; Vector Laboratories, Burlingame, CA, USA), Texas Red-conjugated rabbit-IgG (1:200; Molecular Probe), fluorescein-conjugated mouse IgG (1:200; Molecular Probe), and an OxyBlot protein oxidation detection kit (Chemicon, Temecula, CA). LPS (Sigma) was dissolved 5 μg in 3 μl in sterile phosphate-buffered saline. Fluoxetine was prepared by following the previously reported method (Robertson et al., 1988).

4.2.

Stereotaxic surgery and fluoxetine administration

All experiments were done in accordance with the approved animal protocols and guidelines established by Kyung Hee University. Female Sprague–Dawley (SD) rats (230–260 g) were anesthetized with chloral hydrate (360 mg/kg, intraperitoneal (i.p.) injection) and positioned in a stereotaxic apparatus (Kopf Instrument, Tujunga, CA, USA). Each rat received a unilateral administration of LPS (5 μg in 3 μl PBS) into the right SN [anteroposterior (AP) 5.4 mm, mediolateral (ML) 2.3 mm, and dorsoventral (DV) 7.6 mm from bregma], according to the atlas of Paxinos and Watson (1998). All injections were made using a Hamilton syringe equipped with a 30 S-gauge beveled needle

and attached to a syringe pump (KD Scientific, MA, USA). Infusions were made at a rate of 0.2 μl/min for LPS, or PBS as controls. For histological studies, animals were received i.p. injections of either vehicle (saline) or fluoxetine (5 or 10 mg/kg, twice a day), starting 2 days before LPS injection and continued daily until day 6 post-injection. Animals were humanely killed and their brains harvested at the indicated time points for the various analyses.

4.3.

Tissue preparation and immunohistochemistry

Animals were transcardially perfused with a saline solution containing 0.5% sodium nitrate and heparin (10 U/ml) and fixed with 4% paraformaldehyde dissolved in 0.1 M phosphate buffer (PB). Brains were post-fixed overnight in buffered 4% paraformaldehyde at 4 °C and stored in 30% sucrose solution at 4 °C until they sank, at which time samples were frozensectioned on a sliding microtome. Coronal sections (35 μmthick) were collected in six separate series and processed for immunohistochemical staining as previously described (Choi et al., 2005). In brief, sections were rinsed in PBS then incubated overnight at room temperature (RT) with the following primary antibodies: mouse anti-OX-42 (1:200), mouse anti-ED1 (1:200) for microglia and rabbit anti-tyrosine hydroxylase (TH; 1:2000) for DA neurons, mouse-p67phox (1:200) and rabbit-iNOS (1:400). Stained samples were analyzed under a bright-field microscope (Olympus, Tokyo, Japan) or viewed with a confocal laserscanning microscope (Olympus, Tokyo, Japan).

4.4.

Stereological estimation

The total number of TH-positive neurons was counted in the various animal groups at 7 days post-injection (LPS or PBS) using the optical fractionator method performed on an Olympus CAST (Computer Assisted Stereological Toolbox) system version 2.1.4 (Olympus Denmark A/S, Ballerup, Denmark) as previously described (Choi et al., 2003). This unbiased stereological method of cell counting is not affected by either the reference volume (SNpc) or the size of the counted elements (neurons) (West et al., 1991).

4.5.

In situ detection of O−2 and O−2 -derived oxidants

Hydroethidine histochemistry was performed for in situ visualization of O−2 and O−2-derived oxidants (Choi et al., 2005). Forty-eight hours after LPS injection, hydroethidine (1 mg/ml in PBS containing 1% dimethylsulfoxide; Molecular Probes, Eugene, OR) was administered through tail vein. After 45 min, the animals were transcardially perfused with a saline solution containing 0.5% sodium nitrate and heparin (10 U/ml) and then fixed with 4% paraformaldehyde in 0.1 M PB. After fixation, the brains were cut into 35 μm slices using a sliding microtome. Sections were mounted on gelatin-coated slides, and the oxidized hydroethidine product, ethidium, was examined by confocal microscopy (Olympus Optical).

4.6.

Detection of protein oxidation

As previously described (Choi et al., 2005), the extent of protein oxidation was assessed by measuring protein carbonyl levels

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with an OxyBlot detection kit according to the protocol of the manufacturer with some modifications. Proteins that underwent oxidative modification (i.e., carbonyl group formation) were identified with Western blotting. The optical density of the bands was measured using the Computer Imaging Device and accompanying software (Fujifilm). Levels of protein carbonyls were quantified and expressed as the fold increase versus untreated controls.

4.7.

Appendix A. Supplementary data Supplementary data to this article can be found online at doi:10.1016/j.brainres.2010.09.049.

REFERENCES

Western blot analysis

As previously described (Choi et al., 2005), protein extracts of both the cytosolic and membrane factions were prepared from the ipsilateral SN at 24 h after LPS injection for p67phox analyses. Tissues were gently homogenized using a glass homogenizer in ice-cold buffer consisting of the following (in mM): 20 HEPES, 250 sucrose, 10 KCl, 1.5 MgCl2, 2 EDTA, and protease inhibitor mixture (Sigma). Homogenates were centrifuged for 5 min at 500 ×g at 4 °C, and supernatants were collected and centrifuged for 20 min at 13,000 × g at 4 °C. The pellets were further centrifuged for 1 h at 100,000 × g at 4 °C, and the resulting supernatants and pellets were designated as the cytosolic and membrane fractions, respectively. For iNOS analyses, protein extracts from the ipsilateral SN were prepared as previously described (Choi et al., 2005). Equal amounts of protein (30 μg) were separated by 10% SDS-PAGE, transferred to PVDF membranes using an electrophoretic transfer system, and subjected to immunoblotting with the following specific primary antibodies: mouse-p67phox (1:500) and rabbit-iNOS (1:1000) for overnight at 4 °C. After washing, the membranes were incubated for 1 h at RT with secondary antibodies (1:2000; Amersham Biosciences). Finally, the blots were developed with enhanced chemiluminescence detection reagents (Amersham Biosciences). The blots were subsequently stripped and re-probed with antibodies against mouse-actin (1:5000; Sigma, St. Louis, MO, USA). To determine the relative degree of membrane purification, the membrane fraction was subjected to immunoblotting for calnexin, using a rabbit-calnexin antibody (1:1000; Stressgen, Victoria, British Columbia, Canada). For semiquantitative analyses, the densities of bands on immunoblots were measured with the Computer Imaging Device and accompanying software (Fujifilm). Additionally, levels of p67 in the membrane were expressed as a percentage of the total level of each subunit.

4.8.

149

Statistical analysis

All values are expressed as mean ± SEM. Statistical significance (p < 0.05 for all analyses) was assessed by ANOVA using the Instat 3.05 software package (GraphPad Software, San Diego, CA, USA), followed by Student–Newman–Keuls analyses.

Acknowledgments This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (20090063274).

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