JPET#119206 Edaravone, a Radical Scavenger, Prevents 1-Methyl
JPET Fast Forward. Published on April 11, 2007 as DOI: 10.1124/jpet.106.119206 JPET ThisFast article Forward. has not been Published copyedited andon formatted.April 11, The 2007 final versionas DOI:10.1124/jpet.106.119206 may differ from this version. JPET#119206
Edaravone, a Radical Scavenger, Prevents 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MPTP)-Induced Neurotoxicity in the Substantia Nigra but not the Striatum
Toshiyuki Kawasaki, Kotaro Ishihara, Yukio Ago, Akemichi Baba, and Toshio Matsuda
Laboratory of Medicinal Pharmacology, Graduate School of Pharmaceutical Sciences, Osaka
University, Osaka, Japan (T.K., K.I., Y.A., T.M.); Laboratory of Molecular Neuropharmacology, Downloaded from
Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan (A.B.);
Department of Experimental Disease Model, The Osaka-Hamamatsu Joint Research Center jpet.aspetjournals.org For Child Mental Development, Graduate School of Medicine, Osaka University, Osaka,
Japan (T.M.)
at ASPET Journals on September 28, 2021
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Copyright 2007 by the American Society for Pharmacology and Experimental Therapeutics. JPET Fast Forward. Published on April 11, 2007 as DOI: 10.1124/jpet.106.119206 This article has not been copyedited and formatted. The final version may differ from this version. JPET#119206
Running title: Inhibition by edaravone of MPTP toxicity in substantia nigra
a) Corresponding author: Dr. Toshio Matsuda, Laboratory of Medicinal Pharmacology,
Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita,
Osaka 565-0871, Japan. Tel.: +81 6 6879 8161; Fax: +81 6 6879 8159.
E-mail address: [email protected]
Downloaded from b) The number of text pages: 24
The number of tables: 1 jpet.aspetjournals.org The number of figures: 7
The number of references: 40
The number of words in the Abstract: 229 at ASPET Journals on September 28, 2021
The number of words in Introduction: 484
The number of words in Discussion: 901
A list of nonstandard abbreviations used:
ANOVA, analysis of variance; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrindine; MPP+,
1-methyl-4-phenylpyridinium ion; PBS, phosphate buffer saline; SNc, substantia nigra pars compacta; TBARS, thiobarbituric acid reactive substances. c) Recommended section: Neuropharmacology
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ABSTRACT
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyrindine (MPTP) causes nigrostriatal dopaminergic neurotoxicity and behavioral impairment in rodents, and previous studies suggest that nitric oxide and reactive oxygen species are involved in MPTP-induced neurotoxicity. The present study examines the effect of edaravone, a radical scavenger, on MPTP-induced neurotoxicity in
the striatum and substantia nigra pars compacta (SNc) of C57BL/6J mice. MPTP treatment Downloaded from
(10 mg/kg, s.c. × 4 with 2 h intervals) decreased dopamine levels and tyrosine hydroxylase immunostaining in the striatum and SNc. Pretreatment with edaravone (1 and 3 mg/kg, i.p.) jpet.aspetjournals.org significantly reduced the neurotoxicity in the SNc but not striatum. An immunohistochemical study showed that MPTP caused microglial activation both in the striatum and SNc, while it increased 3-nitrotyrosine immunoreactivity, an in vivo biomarker of peroxynitrite production, at ASPET Journals on September 28, 2021 in the SNc but not the striatum. Furthermore, MPTP increased lipid peroxidation product thiobarbituric acid reactive substance in the midbrain, but not the striatum. Edaravone inhibited activation of the microglia and the increased 3-nitrotyrosine immunoreactivity in the
SNc but not the striatum, and it also inhibited thiobarbituric acid reactive substance levels in the midbrain. Behavioral analyses showed that edaravone improved MPTP-induced impairment of locomotion and rotarod performance. These results suggest that edaravone protects against
MPTP-induced neurotoxicity in the SNc by blocking the production of reactive oxygen species or peroxynitrite, and imply that dopaminergic degeneration in the SNc may play an important role in MPTP-induced motor dysfunction of mice.
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Introduction
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyrindine (MPTP), which impairs mitochondrial respiration by inhibiting complex I, induces suitable models of Parkinson’s disease (Schapira et al., 1990). MPTP causes dopaminergic neurotoxicity, leading to behavioral impairment similar to the features of Parkinson’s disease (Sedelis et al., 2001). Although the mechanisms
of the underlying MPTP-induced dopaminergic neurotoxicity are not completely understood, Downloaded from oxidative stress has been proposed as one of several pathogenic hypotheses (Tieu et al., 2003).
MPTP was found to induce hydroxyl radical formation (Chiueh et al., 1992) and peroxynitrite jpet.aspetjournals.org expression via nitric oxide synthase (Schulz et al., 1995). In fact, MPTP-induced neurotoxicity is ameliorated in transgenic mice with increased superoxide dismutase activity
(Przedborski et al., 1992). Nitric oxide production by neuronal nitric oxide synthase also at ASPET Journals on September 28, 2021 contributes to the MPTP-induced neurotoxicity (Schulz et al., 1995; Przedborski et al., 1996).
On the other hand, it has been reported that activation of microglia may be responsible for
MPTP-induced dopaminergic neurotoxicity (Wu et al., 2002; Wang et al., 2005). The activated microglia can secrete reactive oxygen species, nitric oxide, proinflammatory cytokines, each of which can lead to neuronal damage (Kreutzberg, 1996). Moreover,
Liberatore et al. (1999) reported that MPTP causes upregulation of inducible nitric oxide synthase and that mutant mice lacking the inducible nitric oxide synthase are more resistant to
MPTP than the wild-type littermates. These findings suggest that reactive oxygen species and nitric oxide might participate in the dopaminergic neurotoxicity in MPTP-induced Parkinson’s disease model. In this line, many antioxidants (radical scavengers) such as bromocriptine
(Muralikrishnan and Mohanakumar, 1998), cytosine (Ferger et al., 1998), deprenyl (Ebadi et al.,
2002), and salicylic acid (Mohanakumar et al., 2000) have been reported to protect against
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MPTP-induced neurotoxicity. However, there are discrepant reports concerning the effect of the antioxidant melatonin on MPTP-induced dopaminergic neurotoxicity: some authors have reported that the antioxidant protects against MPTP-induced dopaminergic neurodegeneration
(Acuna-Castroviejo et al., 1997; Antolin et al., 2002; Thomas et al., 2004; Chen et al., 2005), while the others have reported that it does not (Itzhak et al., 1998; Morgan and Nelson, 2001).
Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) is a potent scavenger of hydroxy
radicals and nitric oxide (Satoh et al., 2002), and it has been demonstrated to be beneficial for Downloaded from patients with acute ischemic stroke (Houkin et al., 1998; Yoneda et al., 2003). Bannno et al.
(2005) revealed that edaravone suppressed the production of nitric oxide and reactive oxygen jpet.aspetjournals.org species by activated microglia. We have recently reported that edaravone protected methamphetamine-induced striatal dopaminergic neurotoxicity by scavenging peroxinitrite
(Kawasaki et al., 2006). These findings suggest that edaravone may be effective in improving at ASPET Journals on September 28, 2021 reactive oxygen species- or nitric oxide-mediated neurodegenerative disorders. To further support this idea, we examined the effect of edaravone on MPTP-induced dopaminergic neurotoxicity in the SNc and striatum and MPTP-induced behavioral impairment in C57BL/6J mice. This study shows that the mechanism of MPTP-induced neurotoxicity differs between the SNc and striatum and that edaravone protects against MPTP-induced neurotoxicity in the
SNc but not the striatum.
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Materials and Methods
Animals. Male C57BL/6J 6-week old mice (Japan SLC, Shizuoka, Japan) were housed in cages (24 × 17 × 12 cm) in groups of 5 to 6 under controlled environmental conditions (22 ±
1°C; 12:12 h light-dark cycle, lighting at 08:00 h; food and water ad libitum) for at least 1 week before use in the experiments. The procedures to handle the animals and their care were
conducted according to the Guiding Principles for the Care and Use of Laboratory Animals Downloaded from approved by the Japanese Pharmacological Society.
Drugs and Treatments. The following drugs were used: MPTP (Sigma Aldlich, Osaka, jpet.aspetjournals.org Japan) and edaravone (Mitsubishi Pharma Co., Osaka, Japan). The primary antibodies used were as follows: rabbit polyclonal antibodies against tyrosine hydroxylase; 3-nitrotyrosine
(Chemicon, Temecula, CA, USA); and rabbit polyclonal antibody against ionized at ASPET Journals on September 28, 2021 calcium-binding adapter molecule 1 (Iba-1) (Wako, Osaka, Japan). Vectastain ABC standard kit (Vector Laboratories, Burlingame, CA, USA) was used for immunodetection. All other commercially available chemicals used in the experiments were of superfine quality.
Edaravone was dissolved in 1 N NaOH, adjusted to pH 7.4 with 1 N HCl, and diluted with saline to make 0.2 and 0.6 mg/ml solutions. MPTP was dissolved in saline to make a 2 mg/ml solution. These drugs were injected at a fixed volume of 5 ml/kg body weight. Seven- to
8-week-old mice were subcutaneously administered with MPTP 10 mg/kg four times at 2 h intervals. Edaravone (1 and 3 mg/kg) was intraperitoneally administered 30 min before every
MPTP dosing. The experiments of microglial activation, lipid peroxidation, peroxynitrite production and motor functions were performed one day after the last MPTP dosing, and those of dopamine assay and tyrosine hydroxylase immunostaining were performed 3 days after the last MPTP dosing. Most mice used for behavioral experiments were used for the experiments
6 JPET Fast Forward. Published on April 11, 2007 as DOI: 10.1124/jpet.106.119206 This article has not been copyedited and formatted. The final version may differ from this version. JPET#119206 of microglial activation and peroxynitrite production at 1 day after MPTP and for the experiments for dopamine assay and tyrosine hydroxylase immunostainig at 3 days after MPTP.
Microglial activation and peroxynitrite production were measured in the same mice, and lipid peroxidation was measured in the separate mice.
Measurement of Dopamine Levels. The concentrations of dopamine were quantified by high-performance liquid chromatography (HPLC) with an electrochemical detector (ECD-100;
Eicom, Kyoto, Japan) (Kawasaki et al., 2006). The brains of mice were quickly removed and Downloaded from dissected on an ice-cold glass plate. The striatum and midbrain were individually isolated, frozen on dry ice and stored at −80 °C until assay. Tissue samples were homogenized in 0.2 M jpet.aspetjournals.org perchloric acid containing 100 µM ethylenediaminetetraacetic acid (EDTA) and isoproterenol as an internal standard. The homogenate was centrifuged at 15,000 × g for 15 min at 0°C.
The supernatant was filtered through a 0.22 µm membrane filter (Millipore, Tokyo, Japan), and at ASPET Journals on September 28, 2021 then a 10 µl aliquot of the sample was injected onto the HPLC column every 30 min for dopamine assay. An Eicompak SC-5ODS column (3.0 mm i.d. × 150 mm: Eicom) was used, and the potential of the graphite electrode (Eicom) was set to + 750 mV against an Ag/AgCl reference electrode. The mobile phase contained 0.1 M sodium acetate/0.1 M citrate buffer
(pH 3.5), 190 mg/l octanesulphonic acid, 5 mg/l EDTA, and 17% (vol/vol) methanol. Values are expressed as ng/g or µg/g tissue (wet weight).
Measurement of MPP+ Levels. The 1-methyl-4-phenylpyridinium ion (MPP+) levels were determined according to the HPLC with UV detection method devised by Przedborski et al. (1996). One and 3 h after a single dosing of MPTP (10 mg/kg, s.c.), mice were sacrificed and the striatum and midbrain dissected. Tissue samples were homogenized in 0.1 M perchloric acid containing 100 µM EDTA. After centrifugation (15,000 × g for 15 min at 0°C),
® 10 µl of the supernatant was injected onto a C18-reverse-phase column (Symmetry C18, 5 µm,
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4.6 × 150 mm: Waters). The mobile phase [50 mM potassium phosphate and 11% (vol/vol) acetonitrile] was delivered at a flow rate of 1.0 ml/min. The UV detector was set to 295 nm.
Data were calculated by an external standard calibration. Values are expressed as ng/mg tissue
(wet weight).
Immunohistochemistry. The brains were quickly removed and fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 3 days, and then transferred to 15%
sucrose in 0.1 M phosphate buffer for 2 days. Serial 20-µm-thick coronal sections containing Downloaded from the mid-striatum (+1.2 through −0.1 mm with respect to bregma) and midbrain (−3.4 through
−3.64 mm with respect to bregma) were cut using a cryostat microtome at −20°C. The free jpet.aspetjournals.org floating sections were preincubated for 15 min in 0.3% hydrogen peroxide in 100 mM phosphate buffer saline (PBS) containing 0.3% Triton X-100 (PBS-T). The sections were washed in PBS-T and then incubated with antibodies against tyrosine hydroxylase (1:1000), at ASPET Journals on September 28, 2021
Iba-1 (1:5000) and 3-nitrotyrosine (1:100) in PBS-T overnight at room temperature.
Subsequent primary incubation sections were washed in PBS-T and incubated in a secondary antibody solution containing biotinylated anti-rabbit IgG (Vector, Burlingame, CA) in PBS-T for 2 h at room temperature. The sections were then incubated with the avidin-biotine peroxidase complex (1:2000) in PBS-T for 1 h at room temperature. Visualization was performed using 50 mM Tris-HCl buffer (pH 7.6) containing 0.02% 3,3’-diaminobenzidine and
0.005% hydrogen peroxide with 0.6% nickel ammonium sulfate. The sections were dehydrated with ethanol (70%, 80%, 90%, 100%, xylene) and then mounted and coverslipped.
Immunostaining images in the dorsomedial regions of the mid-striatum sections were collected and analyzed using the NIH Image software package. To quantify the optical density of tyrosine hydroxylase immunostaining and the number of Iba-1-immunoreactive microglia, samples from a 1.0-mm2 area of the dorsomedial regions of the mid-striatum were counted from
8 JPET Fast Forward. Published on April 11, 2007 as DOI: 10.1124/jpet.106.119206 This article has not been copyedited and formatted. The final version may differ from this version. JPET#119206 six independent sections of the right hemisphere, generating an average count for each treated subject. The optical density of tyrosine hydroxylase-positive dopaminergic neurons in the striatum was expressed as percent change from basal levels, with 100% defined as the average count for vehicle treated control mice. The number of tyrosine hydroxylase-immunopositive neurons in the substantia nigra pars compacta (SNc) was quantified by counting blindly from six independent sections of the right hemisphere. To quantify the microglial activation in the
SNc, we used a semi-quantitative scoring system with scores from 1 to 3 to evaluate differences Downloaded from between the treatment groups, as previously reported (Pierri et al., 2005). A blinded observer performed the scoring. Score 1 was given when increased immunoreactivity was observed in jpet.aspetjournals.org the absence of morphologically activated microglia. Score 2 was given when in addition to the criteria for score 1, a number of individually distribution activated microglia were seen. Score
3 was given when in addition to the previous mentioned criteria (scores 1 and 2), clusters of at ASPET Journals on September 28, 2021 activated microglia were observed.
Measurement of Lipid Peroxidation. Lipid peroxidation was assessed by determining the concentrations of thiobarbituric acid reactive substances (TBARS). The TBARS determination was performed spectrophotometrically (Munoz A. et al., 2006). The sample of brain tissue (i.e. striatum or ventral midbrain) was homogenized with three volumes of KCl
(1.15% w/v) containing butylated hydroxytoluene (200 µM). An aliquot of the resulting sample was treated with SDS (8%, w/v) followed by acetic acid (20%), and the mixture was vortexed for 1 min. Thiobarbituric acid (0.8%) was then added and the resulting mixture incubated at 95°C for 60 min. After cooling at room temperature, 3 ml of n-butanol was added and the mixture shaken vigorously. After centrifugation at 1500 × g for 5 min, absorbance of the supernatant (organic layer) was measured at 532 nm using a Shimadzu UV-1650PC spectrophotometer (Kyoto, Japan).
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Rotarod Test. The rotarod (Neuroscience Inc., Tokyo, Japan) consisted of a rotating rod
(diameter 2.8 cm) and individual compartments for each mouse. Mice were trained for two consecutive days prior to MPTP dosing in an acceleration mode (2–16 rpm) for over two minutes. The training was repeated at a fixed speed (16 rpm) until the mice were able to stay on the rod for at least 600 seconds. On day 1 after the MPTP dosing, the mice were assessed for their coordination capability on the rod at 16 rpm for a maximum recording time of 600
seconds. Rotarod test was performed twice every 30 min, and the results were averaged to Downloaded from obtain a single value for each mouse.
Spontaneous Locomotor Activity. Locomotor activity of the mice was measured using a jpet.aspetjournals.org digital counter system with an infrared sensor (Supermex®, Muromachi Kikai, Tokyo, Japan) one day after MPTP dosing. Each mouse was placed in a plastic cage (24 × 17 × 24 cm), and then locomotor activity in each 10 min period was measured for 60 min. at ASPET Journals on September 28, 2021
Statistical Analysis. Data are presented as mean ± SEM or median and range. Data were analyzed using two-way analysis of variance (ANOVA) followed by Tukey–Kramer’s test or the Kruscal–Wallis test followed by a Mann–Whitney U test. Statistical analyses were performed using a software package Statview 5.0J for Apple Macintosh computer (SAS
Institute Inc., Cary, NC, USA). P values of 5% or less were considered statistically significant.
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Results
Effect of Edaravone on MPTP-induced Decrease in Dopamine Levels in the Striatum and Midbrain. Fig. 1 shows the effects of MPTP and edaravone on dopamine levels in the striatum and midbrain of mice. Repeated administration of MPTP (10 mg/kg, four times at 2 h intervals) caused a marked reduction in the striatal dopamine levels 3 days after the last MPTP
dosing. Pretreatment with edaravone (1 and 3 mg/kg) did not attenuate MPTP-induced Downloaded from decrease in the striatal dopamine levels. Two-way ANOVA revealed the main significant effect of MPTP [F (1, 42) = 327.217, P < 0.0001], but not of edaravone [F (2, 42) = 0.054, n.s.]. jpet.aspetjournals.org There was no significant interaction between MPTP and edaravone [F (2, 42) = 0.607, n.s.].
The effect of edaravone against MPTP-induced dopaminergic neurotoxicity in the ventral midbrain was also examined. The dopamine levels in the midbrain were significantly reduced at ASPET Journals on September 28, 2021 in MPTP-treated mice 3 days after the last injection of MPTP. Edaravone at doses of 1 and 3 mg/kg significantly attenuated MPTP-induced reduction in dopamine levels in the midbrain, although edaravone alone did not affect the dopamine levels. Two-way ANOVA revealed the main significant effect of MPTP [F (1, 47) = 24.934, P < 0.0001], but not of edaravone [F (2,
47) = 2.290, n.s.]. There was a significant interaction between MPTP and edaravone [F (2,
47) = 4.939, P = 0.0113].
Effect of Edaravone on MPTP-induced Reduction of Tyrosine Hydroxylase
Immunoreactivity in the Striatum and SNc. We also examined nigrostriatal dopaminergic function by assessing the expression of tyrosine hydroxylase, an immunohistochemical marker for dopaminergic neurons (Fig. 2 and 3). Fig. 2 shows the effects of MPTP and edaravone on tyrosine hydroxylase immunostaining in the striatum. Representative photomicrographs of tyrosine hydroxylase staining in the striatum 3 days after the last MPTP dosing are shown in Fig.
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2A. Fig. 2B shows the densitometric analysis of tyrosine hydroxylase immunostaining in the striatum. Repeated MPTP administration significantly reduced the tyrosine hydroxylase immunostaining in dopaminergic terminals. Pretreatment with edaravone (3 mg/kg) did not affect MPTP-induced reduction of tyrosine hydroxylase immunostaining. Two-way ANOVA revealed the main significant effect of MPTP [F (1, 20) = 269.782, P < 0.0001], but not of edaravone [F (1, 20) = 2.435, n.s.]. There was no significant interaction between MPTP and
edaravone [F (1, 20) = 4.881, n.s.]. Fig. 3 shows the effects of MPTP and edaravone on Downloaded from tyrosine hydroxylase immunostaining in the SNc. Representative photomicrographs of tyrosine hydroxylase staining in the SNc 3 days after the last MPTP dosing are shown in Fig. jpet.aspetjournals.org 3A. Fig. 3B shows the count of tyrosine hydroxylase-immunoreactive neurons in the SNc.
Repeated MPTP administration caused a marked reduction in the number of tyrosine hydroxylase-immunoreactive neurons in the SNc, and this effect was attenuated by at ASPET Journals on September 28, 2021 pretreatment with edaravone (3 mg/kg). Two-way ANOVA revealed the main significant effects of MPTP [F (1, 20) = 131.826, P < 0.0001] and edaravone [F (1, 20) = 17.240, P =
0.0005]. There was a significant interaction between MPTP and edaravone [F (1, 20) =
19.403, P = 0.0003].
Effect of Edaravone on MPTP-induced Microglial Activation in the Striatum and
SNc. Fig. 4 shows the effect of edaravone on MPTP-induced activation of microglia in the striatum of mice. Microglial activation was assessed by staining fixed brain sections with an antibody against Iba-1, a marker of activated microglia. Representative photomicrographs and quantitative results of Iba-1 immunostaining in the striatum one day after the last MPTP dosing are shown (Fig. 4A). Edaravone (3 mg/kg) alone did not affect the number of activated microglia. Repeated MPTP administration caused a robust increase in the number of activated microglia. Two-way ANOVA revealed the main significant effect of MPTP [F (1, 20) =
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176.977, P < 0.0001], but not of edaravone [F (1, 20) = 2.023, n.s.] (Fig. 4B). Pretreatment with edaravone (3 mg/kg) did not affect MPTP-induced activation of microglia in the striatum
[F (1, 20) = 0.943, n.s.]. Fig. 5 shows the effect of edaravone on MPTP-induced activation of microglia in the SNc. Representative photomicrographs of Iba-1 immunostaining are shown in Fig. 5A. We observed changes in cellular morphology, such as enlarged cell bodies and thickening of their processes. The microglial response was evaluated by the semi-quantitative
scoring system described by Pierri et al. (2005) (Fig. 5B), since it was difficult to count Downloaded from activated microglia in the SNc. Kruscal–Wallis test revealed the significant effect of the treatment [P < 0.001]. Few morphologically activated microglia were expressed in jpet.aspetjournals.org vehicle-treated mice (median, 1; range, 1 to 2). Administration of MPTP displayed clusters of activated microglia in the SNc (median, 3; range, 2 to 3) [P < 0.01 versus vehicle treated controls]. Pretreatment with edaravone (3 mg/kg) attenuated significantly the MPTP-induced at ASPET Journals on September 28, 2021 activation of microglia in the SNc (median, 1.5; range, 1 to 2) [P < 0.01 versus MPTP treated controls]. Edaravone (3 mg/kg) alone did not cause a significant change in the morphology of the microglia (median, 1; range, 1 to 2).
Effect of Edaravone on MPTP-induced Lipid Peroxidation and Peroxynitrite
Production. Fig. 6 shows the effect of edaravone on MPTP-induced lipid peroxidation and peroxynitrite production in mouse brain. The level of TBARS is an index of lipid peroxidation
(Fig. 6A) and formation of 3-nitrotyrosine is an index of peroxynitrite production (Fig. 6B).
Both indices are indicated as markers of oxidative stress status. Repeated MPTP administration induced a marked increase in the production of TBARS in the ventral midbrain
[F (1, 20) = 23.076, P = 0.0001], but not the striatum [F (1, 20) = 1.361, n.s.] one day after the last injection of MPTP. Edaravone (3 mg/kg) significantly inhibited the TBARS production in the ventral midbrain. Two-way ANOVA revealed a significant interaction between MPTP and
13 JPET Fast Forward. Published on April 11, 2007 as DOI: 10.1124/jpet.106.119206 This article has not been copyedited and formatted. The final version may differ from this version. JPET#119206 edaravone [F (1, 20) = 15.128, P = 0.0009]. The 3-nitrotyrosine immunoreactivity was markedly increased in the SNc by MPTP treatment (Fig. 6B-e), while MPTP did not affect the
3-nitrotyrosine immunoreactivity in the striatum (Fig. 6B-b). Pretreatment with edaravone of
3 mg/kg inhibited the effect of MPTP on 3-nitrotyrosine formation in the SNc (Fig. 6B-f). In addition, edaravone alone did not alter 3-nitrotyrosine immunoreactive levels in the striatum and SNc (data not shown).
Effects of Edaravone on MPTP-induced Behavioral Deficits. Fig. 7A shows the effect Downloaded from of edaravone on MPTP-induced locomotor deficits in a novel environment. Administration of
MPTP significantly reduced spontaneous locomotor activity in mice [F (1, 92) = 26.496, P < jpet.aspetjournals.org 0.0001]. Pretreatment with edaravone (3 mg/kg) significantly attenuated MPTP-induced hypolocomotion [F (2, 92) = 5.028, P = 0.0085], while edaravone alone did not alter basal motor activity [F (2, 92) = 2.682, n.s.]. Fig. 7B shows the effect of edaravone on at ASPET Journals on September 28, 2021
MPTP-induced loss of motor coordination in the rotarod test. Rotarod test is widely used to measure coordinated motor skills that have been employed in the MPTP mouse model (Sedelis et al., 2001). All mice were trained and were able to stay on the rotating rod at a fixed speed of
16 rpm for 600 seconds. One day after MPTP dosing, the mice spent significantly less time overall on the rotating rod, indicating a loss of motor coordination [F (1, 87) = 8.208, P =
0.0052]. This rotarod performance was improved by edaravone at doses of 1 and 3 mg/kg.
Two-way ANOVA revealed a significant interaction between MPTP and edaravone [F (2, 87) =
8.739, P = 0.0003]. Edaravone alone did not affect rotarod performance.
Effect of Edaravone on MPP+ Levels in the Brain. To exclude the pharmacokinetic effects of edaravone due to differences in MPTP metabolism, uptake or elimination, we measured the MPP+ levels at different time points after a single administration of MPTP (10 mg/kg) (Table 1). Edaravone (3 mg/kg) did not affect the MPP+ levels in these regions at 1 and
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3 h after treatment. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 28, 2021
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Discussion
The present study demonstrates that MPTP caused dopaminergic neurodegeneration in the
SNc and striatum. MPTP decreased dopamine levels and tyrosine hydroxylase immunostaining in the SNc and striatum and increased Iba-I immunoreactivity, a marker of activated microglia. Although the degrees of decrease in tyrosine hydroxylase
immunostaining and activation of microglia were similar between the SNc and striatum, the Downloaded from degree of decrease in dopamine levels was higher in the striatum than the midbrain including
SNc. In this regard, Kurosaki et al. (2004) reported that MPTP-induced decrease in tyrosine jpet.aspetjournals.org hydroxylase immunostaining was observed in the striatum faster than the SNc. These results suggest that MPTP causes degenerative loss of nigrostriatal dopamine neurons and the progression of striatal damage worsens to that of nigral damage after MPTP treatment. MPTP at ASPET Journals on September 28, 2021 increased TBARS levels and 3-nitrotyrosine immunoreactivity, a marker of peroxynitrite, in the
SNc but not the striatum. These changes in the SNc were reduced by the radical scavenger edaravone. In addition, edaravone attenuated decreases in dopamine levels and tyrosine hydroxylase immunostaining in the SNc but not the striatum. These findings suggest that reactive oxygen species and nitric oxide play a key role in MPTP-induced dopaminergic degeneration in the SNc, but not in the striatum. The finding that edaravone protects against only the nigral neurotoxicity appears to contrast with the previous observation that the drug blocks the striatal neurotoxicity in methamphetamine-treated mice (Kawasaki et al., 2006).
This may be explained by the difference in the mechanism for neurotoxicity between methamphetamine and MPTP models. We have found in the separate experiments that methamphetamine increased TBARS levels in the striatum and edaravone blocked methamphetamine-induced increases in TBARS levels and 3-nitrotyrosine immunoreactivity.
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Regarding MPTP-induced striatal neurotoxicity, Sriram et al. (2006) reported that tumor necrosis factor-α may play a role in MPTP- or methamphetamine-induced selective degeneration of striatal dopaminergic nerve terminals. The exact mechanism for
MPTP-induced neurodegeneration in the SNc and striatum remains to be determined. It should be noted that edaravone attenuated microglial activation in the SNc but not the striatum.
In this regard, Hayley et al. (2004) reported that Fas deficiency attenuated MPTP-induced
microglial activation in the SNc but not the striatum. These results suggest that MPTP causes Downloaded from microglial activation via different mechanisms in the SNc and striatum.
There are conflicting results on the effect of the antioxidant, melatonin, on MPTP-induced jpet.aspetjournals.org neurotoxicity. The neuroprotective effect of melatonin was demonstrated in the SNc (Antolin et al., 2002; Thomas et al., 2004; Chen et al., 2005), while the result was negative in the striatum (Van der Schyf at al., 2000; Morgan and Nelson, 2001; Itzhak et al., 1998). Only one at ASPET Journals on September 28, 2021 report has shown that melatonin has a protective effect against MPTP-induced neurotoxicity both in the SNc and striatum, but the degree of the protective effect in the striatum was small (Li et al., 2002). The discrepancy may be explained by the difference in the brain regions. This point may be explained by the present finding that the effect of edaravone on MPTP-induced neurotoxicity differs between the SNc and striatum. Edaravone protected MPTP-induced decreases in tyrosine hydroxylase immunostaining in the SNc and dopamine levels in the midbrain, but had no effect on those in the striatum. It should be noted that edaravone did not affect MPP+ levels in the striatum and SNc. This suggests that the effect of edaravone is not related to the metabolism of MPTP.
Motor impairment is observed only after severe dopamine depletion (70-80%) in the striatum in Parkinson’s disease patients (Riederer and Wuketich, 1976). This is a basis for dopamine replacement therapies in Parkinson’s disease. The sites of action of L-dopa are not
17 JPET Fast Forward. Published on April 11, 2007 as DOI: 10.1124/jpet.106.119206 This article has not been copyedited and formatted. The final version may differ from this version. JPET#119206 only striatal dopaminergic terminals, but also the SNc (Robertson and Robertson., 1989;
Crocker, 1997; Fox et al., 1998). Previous studies indicate the importance of dopaminergic function in the SNc being preserved in the improvement of MPTP-induced motor dysfunction.
Crocker et al. (2003) showed that calpain inhibition attenuated MPTP-induced locomotor deficits without recovering striatal dopamine levels. In addition, Hayley et al. (2004) demonstrated that Fas deficiency attenuated MPTP-induced dopaminergic loss and microglial
activation in the SNc but not the striatum. In view of the evidence that nigral dopaminergic Downloaded from neurons release dopamine not only from their axons projecting to the striatum but also their dendrites (Bjorklund and Lindvall, 1975; Cheramy et al., 1981), Park’s group (Crocker et al., jpet.aspetjournals.org 2003; Hayley et al., 2004) hypothesized that preservation of the SNc may contribute to improving functional outcome through stimulation of dopamine turnover in the striatum and extrastriatal regulation of the basal ganglia. The hypothesis is consistent with the present at ASPET Journals on September 28, 2021 finding that edaravone improves MPTP-induced behavioral changes (hypolocomotion and impaired rotarod performance) and protects against MPTP-induced neurotoxicity in the SNc but not the striatum. Then, it is likely that that dopaminergic activity in the SNc plays a key role in edaravone-induced improvement of MPTP-induced motor dysfunction.
In conclusion, the present study shows that MPTP increases the production of reactive oxygen species or nitric oxide and causes microglial activation in the SNc but not the striatum.
In addition, the radical scavenger edaravone protects against MPTP-induced neurodegeneration in the SNc but not the striatum and improves MPTP-induced motor dysfunction. The present study in a MPTP-induced dopaminergic degeneration model, together with the recent finding in a methamphetamine-induced model (Kawasaki et al., 2006), suggest that edaravone may be useful for treating Parkinson’s disease and other oxidative damage-related neurodegenerative diseases, although the drug is used for the treatment of acute stroke (Houkin et al., 1998).
18 JPET Fast Forward. Published on April 11, 2007 as DOI: 10.1124/jpet.106.119206 This article has not been copyedited and formatted. The final version may differ from this version. JPET#119206
References
Acuna-Castroviejo D, Cotomontes A, Monti GG, Oritis GG, and Reiter RJ (1997) Melatonin is
protective against MPTP-induced striatal and hippocampal lesions. Life Sci 60:23-29.
Antolin I, Mayo JC, Sainz RM, del Brio Mde L, Herrera F, Martin V, and Rodriguez C (2002)
Protective effect of melatonin in a chronic experimental model of Parkinson's disease. Brain
Res 943:163-173. Downloaded from
Banno M, Mizuno T, Kato H, Zhang G, Kawanokuchi J, Wang J, Kuno R, Jin S, Takeuchi H,
and Suzumura A (2005) The radical scavenger edaravone prevents oxidative neurotoxicity jpet.aspetjournals.org induced by peroxynitrite and activated microglia. Neuropharmacology 48:283-290.
Bjorklund A, and Lindvall O (1975) Dopamine in dendrites of substantia nigra neurons:
suggestions for a role in dendritic terminals. Brain Res 83:531-537. at ASPET Journals on September 28, 2021
Chen LJ, Gao YQ, Li XJ, Shen DH, and Sun FY (2005) Melatonin protects against
MPTP/MPP+-induced mitochondrial DNA oxidative damage in vivo and in vitro. J Pineal
Res 39:34-42.
Cheramy A, Leviel V, and Glowinski J (1981) Dendritic release of dopamine in the substantia
nigra. Nature 289:537-542.
Chiueh CC, Krishna G, Tulsi P, Obata T, Lang K, Huang S-L, and Murphy DL (1992)
Intracranial microdialysis of salicylic acid to detect hydroxyl radical generation through
dopamine autoxidation in the caudate nucleus: effect of MPP+. Free Radic. Biol. Med.
13:581-583.
Crocker AD (1997) The regulation of motor control: an evaluation of the role of dopamine
receptors in the substantia nigra. Rev Neurosci 8:55-76.
Crocker SJ, Smith PD, Jackson-Lewis V, Lamba WR, Hayley SP, Grimm E, Callaghan SM,
19 JPET Fast Forward. Published on April 11, 2007 as DOI: 10.1124/jpet.106.119206 This article has not been copyedited and formatted. The final version may differ from this version. JPET#119206
Slack RS, Melloni E, Przedborski S, Robertson GS, Anisman H, Merali Z, and Park DS
(2003) Inhibition of calpains prevents neuronal and behavioral deficits in an MPTP mouse
model of Parkinson's disease. J Neurosci 23:4081-4091.
Ebadi M, Sharma S, Sjavali S, and El Refaey H (2002) Neuroprotective actions of selegiline. J
Neurosci Res 67:285-289.
Ferger B, Spratt C, Teismann P, Seitz G, and Kuschinsky K (1998) Effects of sytisine on
hydroxyl radicals in vitro and MPTP-induced dopamine depletion in vivo. Eur J Pharmacol Downloaded from
360:155-163.
Fox SH, Moser B, and Brotchie JM (1998) Behavioral effects of 5-HT2C receptor antagonism in jpet.aspetjournals.org the substantia nigra zona reticulata of the 6-hydroxydopamine-lesioned rat model of
Parkinson’s disease. Exp Neurol 151:35-49.
Hayley S, Crocker SJ, Smith PD, Jackson-Lewis V, Przedborski S, Mount M, Slack R, Anisman at ASPET Journals on September 28, 2021
H, and Park DS (2004) Regulation of dopaminergic loss by Fas in a
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson’s disease. J Neurosci
24:2045-2053.
Houkin K, Nakayama N, Kamda K, Noujou T, Abe H, and Kashiwaba T (1998)
Neuroprotective effects of the free radical scavenger MCI-186 in patients with cerebral
infarction: clinical evaluation using magnetic resonance imaging and spectroscopy. J Stroke
Cerebrovas Dis 7:315-322.
Itzhak Y, Martin JL, Black MD, and Ali SF (1998) Effect of melatonin on methamphetamine-
and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced dopaminergic neurotoxicity and
methamphetamine-induced behavioral sensitization. Neuropharmacology 37:781-791.
Kawasaki T, Ishihara K, Ago Y, Nakamura S, Itoh S, Baba A, and Matsuda T (2006) Protective
effect of the radical scavenger edaravone against methamphetamine-induced dopaminergic
20 JPET Fast Forward. Published on April 11, 2007 as DOI: 10.1124/jpet.106.119206 This article has not been copyedited and formatted. The final version may differ from this version. JPET#119206
neurotoxicity in mouse striatum. Eur J Pharmacol 542:92-99.
Kreutzberg GW (1996) Microglia: a sensor for pathological events in the CNS. Trends Neurosci
19:312-318.
Kurosaki R, Muramatsu Y, Kato H, and Araki T (2004) Biochemical, behavioral and
immunohistochemical alterations in MPTP-treated mouse model of Parkinson’s disease.
Pharmacol Biochem Behav 78:143-153.
Li XJ, Gu J, Lu SD, and Sun FY (2002) Melatonin attenuates MPTP-induced dopaminergic Downloaded from
neuronal injury associated with scavenging hydroxyl radical. J Pineal Res 32:47-52.
Liberatore GT, Jackson-Lewis V, Vukosavic S, Mandir AS, Vila M, McAuliffe WG, Dawson jpet.aspetjournals.org VL, Dawson TM, and Przedborski S (1999) Inducible nitric oxide synthase stimulates
dopaminergic neurodegeneration in the MPTP model of Parkinson disease. Nat Med
5:1403-1409. at ASPET Journals on September 28, 2021
Mohanakumar KP, Muralikrishnan D, and Thomas B (2000) Neuroprotection by sodium
salicylate against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurotoxicity.
Brain Res 864:281-290.
Morgan WW, and Nelson JF (2001) Chronic administration of pharmacological levels of
melatonin does not ameliorate the MPTP-induced degeneration of the nigrostriatal pathway.
Brain Res 921:115-121.
Munoz A, Rey P, Guerra MJ, Mendez-Alvarez E, Soto-Otero R, and Labandeira-Garcia JL
(2006) Reduction of dopaminergic degeneration and oxidative stress by inhibition of
angiotensin converting enzyme in a MPTP model of parkinsonism. Neuropharmacology
[Epub ahead of print]
Muralikrishnan D, and Mohanakumar KP (1998) Neuroprotection by bromocriptine against
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurotoxicity in mice. FA S E B J
21 JPET Fast Forward. Published on April 11, 2007 as DOI: 10.1124/jpet.106.119206 This article has not been copyedited and formatted. The final version may differ from this version. JPET#119206
12:905-912.
Pierri M, Vaudano E, Sager T, and Englund U (2005) KW-6002 protects from MPTP induced
dopaminergic toxicity in the mouse. Neuropharmacology 48:517-524.
Przedborski S, Kostic V, Jackson-Lewis V, Naini AB, Simonetti S, Fahn S, Carlson E, Epstein
CJ, and Cadet JL (1992) Transgenic mice with increased Cu/Zn-superoxide dismutase
activity are resistant to N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced
neurotoxicity. J Neurosci 12:1658-1667. Downloaded from
Przedborski S, Jackson-Lewis V, Yokoyama R, Shibata T, Dawson VL, and Dawson TM (1996)
Role of neuronal nitric oxide in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine jpet.aspetjournals.org (MPTP)-induced dopaminergic neurotoxicity. Proc Natl Acad Sci USA 93:4565-4571.
Riederer P, and Wuketich S (1976) Time course of nigrostriatal degeneration in Parkinson's
disease. A detailed study of influential factors in human brain amine analysis. J Neural at ASPET Journals on September 28, 2021
Transm 38:277-301.
Robertson GS, and Robertson HA (1989) Evidence that L-dopa-induced rotational behavior is
dependent on both striatal and nigral mechanisms. J Neurosci 9:3326-3331.
Satoh K, Ikeda Y, Shioda S, Tobe T, and Yoshikawa T (2002) Edarabone scavenges nitric oxide.
Redox Rep 7:219-222.
Sedelis M, Schwarting RK, and Huston JP (2001) Behavioral phenotyping of the MPTP mouse
model of Parkinson's disease. Behav Brain Res 125:109-125.
Schapira AH, Cooper JM, Dexter D, Clark JB, Jenner P, and Marsden CD (1990) Mitochondrial
complex I deficiency in Parkinson's disease. J Neurochem 54:823-827.
Schulz JB, Matthews RT, Muqit MM, Browne SE, and Beal MF (1995) Inhibition of neuronal
nitric oxide synthase by 7-nitroindazole protects against MPTP-induced neurotoxicity in
mice. J Neurochem 64:936-939.
22 JPET Fast Forward. Published on April 11, 2007 as DOI: 10.1124/jpet.106.119206 This article has not been copyedited and formatted. The final version may differ from this version. JPET#119206
Sriram K, Miller DB, and O'callaghan JP (2006) Minocycline attenuates microglial activation
but fails to mitigate striatal dopaminergic neurotoxicity: role of tumor necrosis factor-alpha.
J Neurochem 96:706-718.
Thomas B, and Mohanakumar KP (2004) Melatonin protects against oxidative stress caused by
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in the mouse nigrostriatum. J Pineal Res
36:25-32.
Tieu K, Ischiropoulos H, and Przedborski S (2003) Nitric oxide and reactive oxygen species in Downloaded from
Parkinson's disease. IUBMB Life 55:329-335.
Van der Schyf, CJ, Castagnoli K, Palmer S, Hazelwood L, and Castagnoli JrN (2000) Melatonin jpet.aspetjournals.org fails to protect against long-term MPTP-induced dopamine depletion in mouse striatum.
Neurotoxicity Res 1:261-269.
Wang T, Pei Z, Zhang W, Liu B, Langenbach R, Lee C, Wilson B, Reece JM, Miller DS, Hong at ASPET Journals on September 28, 2021
JS (2005) MPP+-induced COX-2 activation and subsequent dopaminergic
neurodegeneration. FASEB J 19:1134-1136.
Wu DC, Jackson-Lewis V, Vila M, Tieu K, Teismann P, Vadseth C, Choi DK, Ischiropoulos H,
and Przedborski S (2002) Blockade of microglial activation is neuroprotective in the
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson disease. J
Neurosci 22:1763-1771.
Yoneda Y, Uehara T, Yamasaki H, Kita Y, Tabuch M, and Mori E (2003) Hospital-based study
of the care and cost of acute ischemic stroke in Japan. Stroke 34:718-724.
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Footnotes
This work was supported in part by grants from the Ministry of Education, Science, Sports and Culture of Japan, and from Mitsubishi Pharma Corporation.
Downloaded from jpet.aspetjournals.org at ASPET Journals on September 28, 2021
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Legends for Figures
Fig. 1. Effect of edaravone on MPTP-induced depletion of dopamine levels in the striatum and midbrain. Mice were administered 10 mg/kg of MPTP s.c. four times at 2 h intervals.
Vehicle or edaravone at doses of 1 and 3 mg/kg was administered i.p. 30 min before every
MPTP dosing. Mice were sacrificed 3 days after the last MPTP dosing for the assessment of
dopamine levels in the striatum and midbrain. n: the number of mice used. Results are Downloaded from expressed as the mean ± SEM of 6 to 11 mice. **: P < 0.01, compared with the vehicle/vehicle treatment group; ##: P < 0.01, compared with the MPTP/vehicle treatment group. jpet.aspetjournals.org
Fig. 2. Effect of edaravone on MPTP-induced decrease in the immunoreactivity for tyrosine hydroxylase in the striatum. Mice were administered 10 mg/kg of MPTP s.c. four times at 2 h at ASPET Journals on September 28, 2021 intervals. Vehicle or edaravone of 3 mg/kg was administered i.p. 30 min before every MPTP dosing. Mice were sacrificed 3 days after the last MPTP dosing. (A) Representative photomicrographs of tyrosine hydroxylase staining in the striatum are shown. Scale bar = 1 mm. (B) Tyrosine hydroxylase immunostaining in the striatum was quantified in each group.
Densitometirc analysis using NIH Image software package for the tyrosine hydroxylase-positive fibers is shown. Results are expressed as the mean ± SEM of 6 mice.
**: P < 0.01, compared with the vehicle/vehicle treatment group.
Fig. 3. Effect of edaravone on MPTP-induced decrease in the immunoreactivity for tyrosine hydroxylase in the substantia nigra. Mice were administered 10 mg/kg of MPTP s.c. four times at 2 h intervals. Vehicle or edaravone of 3 mg/kg was administered i.p. 30 min before every MPTP dosing. Mice were sacrificed 3 days after the last MPTP dosing. (A)
25 JPET Fast Forward. Published on April 11, 2007 as DOI: 10.1124/jpet.106.119206 This article has not been copyedited and formatted. The final version may differ from this version. JPET#119206
Representative photomicrographs of tyrosine hydroxylase staining in the SNc are shown.
Scale bar = 200 µm. (B) Tyrosine hydroxylase immunostaining in the SNc was quantified in each group. The number of tyrosine hydroxylase-immunopositive neurons is shown. Results are expressed as the mean ± SEM of 6 mice. **: P < 0.01, compared with the vehicle/vehicle treatment group; ##: P < 0.01, compared with the MPTP/vehicle treatment group.
Fig. 4. Effect of edaravone on MPTP-induced activation of microglia in the striatum. Mice Downloaded from were administered with 10 mg/kg of MPTP s.c. four times at 2 h intervals. Vehicle or edaravone of 3 mg/kg was administered i.p. 30 min before every MPTP dosing. Mice were jpet.aspetjournals.org sacrificed 1 day after the last MPTP dosing. (A) Representative photomicrographs of Iba-1 immunostaining in the striatum are shown. Scale bar = 100 µm. (B) Quantitative results of
Iba-1 immunostaining in each group are shown. Results are expressed as the mean ± SEM of 6 at ASPET Journals on September 28, 2021 mice. **: P < 0.01, compared with the vehicle/vehicle treatment group.
Fig. 5. Effect of edaravone on MPTP-induced activation of microglia in the substantia nigra.
Mice were administered with 10 mg/kg of MPTP s.c. four times at 2 h intervals. Vehicle or edaravone of 3 mg/kg was administered i.p. 30 min before every MPTP dosing. Mice were sacrificed 1 day after the last MPTP dosing. (A) Representative photomicrographs of Iba-1 immunostaining in the SNc are shown. Scale bar = 200 µm; 100 µm in inset. (B) Qualitative results of Iba-1 immunostaining in each group are shown. The microglial activation in the
SNc was assessed using microglia score (see in the Materials and Methods). The horizontal lines represent median values. **: P < 0.01, compared with the vehicle/vehicle treatment group; ##: P < 0.01, compared with the MPTP/vehicle treatment group.
26 JPET Fast Forward. Published on April 11, 2007 as DOI: 10.1124/jpet.106.119206 This article has not been copyedited and formatted. The final version may differ from this version. JPET#119206
Fig. 6. Effect of edaravone on MPTP-induced lipid peroxidation and peroxynitrite production in the mouse brain. Mice were administered with 10 mg/kg of MPTP s.c. four times at 2 h intervals. Vehicle or edaravone of 3 mg/kg was administered i.p. 30 min before every MPTP dosing. Mice were sacrificed 1 day after the last MPTP dosing. Lipid peroxidation (A) was assessed by the formation of TBARS in the striatum and ventral midbrain. Results are expressed as the mean ± SEM of 6 mice. **: P < 0.01, compared with the vehicle/vehicle
treatment group; ##: P < 0.01, compared with the MPTP/vehicle treatment group. Downloaded from
Representative photomicrographs of 3-nitrotyrosine immunostaining (B) in the striatum (a-c) and SNc are shown (d-f). Scale bar = 200 µm; 100 µm in inset. Results are representative of jpet.aspetjournals.org 3 independent experiments.
Fig. 7. Effect of edaravone on MPTP-induced behavioral deficits in mice. Mice were at ASPET Journals on September 28, 2021 administered with 10 mg/kg of MPTP s.c. four times at 2 h intervals. Vehicle or edaravone at doses of 1 and 3 mg/kg was administered i.p. 30 min before every MPTP dosing. One day after the last MPTP dosing, spontaneous locomotor activity in a novel environment (A) and motor coordination on the rotating rod (B) were assessed. n: the number of mice used.
Results are expressed as the mean ± SEM of 7 to 23 mice. **: P < 0.01, compared with the vehicle/vehicle treatment group; ##: P < 0.01, compared with the MPTP/vehicle treatment group.
27 JPET Fast Forward. Published on April 11, 2007 as DOI: 10.1124/jpet.106.119206 This article has not been copyedited and formatted. The final version may differ from this version. JPET#119206
Table 1 Effect of edaravone on MPP+ levels in the striatum and ventral midbrain of mice.
The MPP+ contents in the striatum and ventral midbrain were measured 1 and 3 h after a single injection of MPTP (10 mg/kg, s.c.). Vehicle or edaravone 3 mg/kg was administered i.p. 30 min before the MPTP injection. Results are expressed as the mean ± SEM of 4 mice.
Treatment MPP+ levels (ng/mg wet tissue weight) Downloaded from Striatum Midbrain 1 h 3 h 1 h 3 h MPTP + vehicle 7.26 ± 0.42 2.26 ± 0.11 3.91 ± 0.48 1.86 ± 0.09 MPTP + edaravone 7.39 ± 0.69 2.19 ± 0.14 4.00 ± 0.48 1.78 ± 0.14 jpet.aspetjournals.org
at ASPET Journals on September 28, 2021
28 JPET Fast Forward. Published on April 11, 2007 as DOI: 10.1124/jpet.106.119206 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 28, 2021 JPET Fast Forward. Published on April 11, 2007 as DOI: 10.1124/jpet.106.119206 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 28, 2021 JPET Fast Forward. Published on April 11, 2007 as DOI: 10.1124/jpet.106.119206 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 28, 2021 JPET Fast Forward. Published on April 11, 2007 as DOI: 10.1124/jpet.106.119206 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 28, 2021 JPET Fast Forward. Published on April 11, 2007 as DOI: 10.1124/jpet.106.119206 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 28, 2021 JPET Fast Forward. Published on April 11, 2007 as DOI: 10.1124/jpet.106.119206 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 28, 2021 JPET Fast Forward. Published on April 11, 2007 as DOI: 10.1124/jpet.106.119206 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 28, 2021