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Neuroprotective effects of 3-O-demethylswertipunicoside against MPTP-induced Parkinson׳s disease in vivo and its antioxidant properties in vitro
brain research 1624 (2015) 78–85
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Research report
Neuroprotective effects of 3-O-demethylswertipunicoside against MPTP-induced Parkinson's disease in vivo and its antioxidant properties in vitro Jun-Jun Zhoua,b,c, Shen-Yu Zhaia,c, Hui-Nan Zhanga,c, Yue-Hua Wangc,d, Xiao-Ping Pua,c,n a
National Key Research Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, PR China Department of Pharmacology, Dalian Medical University, Dalian 116044, PR China c Department of Molecular and Cellular Pharmacology, School of Pharmaceutical Sciences, Peking University, Beijing 100191, PR China d Beijing Key Laboratory of Drug Target Identification and Drug Screening, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, PR China b
ar t ic l e in f o
abs tra ct
Article history:
3-O-demethylswertipunicoside (3-ODS) has been reported to protect dopaminergic neurons
Accepted 30 June 2015
against neurotoxicity induced by 1-methyl-4-phenylpyridinium (MPPþ) in PC12 cells. Here, we
Available online 23 July 2015
investigate the neuroprotective effects in vivo and antioxidant activities in vitro of 3-ODS. In the 1-
Keywords:
methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP)-treated mouse model of Parkinson's disease
3-O-demethylswertipunicoside
(PD), 3-ODS dose-dependently improved motor coordination (as shown by rotarod test),
1-methyl-4-phenyl-1, 2, 3
increased the contents of dopamine (DA) and its metabolites in the striatum, and increased
6-tetrahydropyridine
the number of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra (SN). In
Parkinson's disease
addition, 3-ODS also increased the spine density in hippocampal CA1 neurons. In antioxidant
Neuroprotection
assays, 3-ODS showed a strong capacity in scavenging hydroxyl radical, superoxide anion and 1,
Growth-promoting effect
1-diphenyl-2-picrylhydrazyl (DPPH) radical in a concentration-dependent manner. Taken
Antioxidant activity
together, we conclude that 3-ODS attenuates the PD-related motor deficits mainly through its neuroprotective effects, growth-promoting effects on spine density, and its antioxidant activities. & 2015 Elsevier B.V. All rights reserved.
1.
Introduction
neurons in the substantia nigra (SN) (Hirsch et al., 1988). However, the mechanism of dopaminergic neurodegenera-
Parkinson's disease (PD) is a common neurodegenerative disease characterized by progressive loss of dopaminergic
tion is still poorly understood. The neurotoxin 1-methyl-4phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) is a mitochondrial
n Corresponding author at: National Key Research Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, PR China. Fax: þ86 10 8280 2431. E-mail address: [email protected] (X.-P. Pu).
http://dx.doi.org/10.1016/j.brainres.2015.06.051 0006-8993/& 2015 Elsevier B.V. All rights reserved.
brain research 1624 (2015) 78–85
complex I inhibitor that can cross the blood-brain barrier and be converted to its toxic metabolite 1-methyl-4phenylpyridinium (MPPþ), which selectively damages dopaminergic neurons and induces DA depletion (Anderson et al., 2006; Dauer and Przedborski, 2003). MPTP-induced parkinsonian mice provide one of the most commonly used animal models for analyzing the effects of many compounds that act on dopaminergic neurons (Patil et al., 2014; Tomac et al., 1995). It is widely accepted that oxidative stress caused by toxic free radicals plays an important role in the pathogenesis of various neurodegenerative diseases such as PD (Dexter et al., 1989; Halliwell et al., 1992; Reed, 2011). Therefore, reducing such free radicals has become a central focus of research designed to prevent or ameliorate neurodegeneration. Antioxidants from natural products or medicinal plants are being to emerge as candidates for prevention or treatment of neurodegenerative diseases. Swertia punicea HEMSL (Gentianaceae) is a traditional Chinese medicine with numerous therapeutic applications, including treatment of hepatitis, cholecystitis, and urinary tract infection (Zheng et al., 2014; Zhong et al., 2010). The xanthone compound 3-O-demethylswertipunicoside (3-ODS) is extracted from S. punicea (Du et al., 2010; Tan et al., 1992). Our previous studies have shown that 3-ODS (Fig. 1) has a neuroprotective effect against H2O2 and MPPþ-induced oxidative stress and apoptosis in PC12 cells. The mechanisms of 3ODS may involve antioxidative defense and anti-apoptotic activities (Zhang et al., 2010; Zhou et al., 2013). The aim of this study was to evaluate the neuroprotective effects and mechanisms of 3-ODS using an in vivo MPTPinduced mouse model of PD in C57 BL/6 mice and ex vivo hippocampal slice of rats. We have also investigated the possible antioxidant activities of 3-ODS using various free radical scavenging tests in vitro, such as hydroxyl radical scavenging, superoxide radical scavenging and 1, 1-diphenyl2-picryl-hydrazyl (DPPH) radical scavenging.
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the control group (Po0.01). Pretreated with higher doses of 3ODS (50 and 100 mg/kg) significantly attenuated the reduction in the latent period (both Po0.01). Pargyline (15 mg/kg), as the positive control group, also increased the latent period compared with the MPTP-treated group (Po0.01).
2.2. Effects of 3-ODS on the levels of dopamine (DA) and 3,4-dihydroxyphenyl acetic acid (DOPAC) in the striatum The effects of 3-ODS on the levels of DA and its metabolite DOPAC in the striatum are shown in Fig. 3. ANOVA revealed significant differences between all groups (F¼ 35.26, Po0.0001; F¼ 13.61, Po0.0001) for DA and DOPAC contents, respectively. DA and DOPAC contents in the MPTP-treated group were significantly lower than those in the control group (both Po0.01). After administration of medium to high doses of 3-ODS (50 and 100 mg/kg), the striatal DA contents were substantially rescued (both Po0.01). For the striatal DOPAC content, only the low-dose 3-ODS (25 mg/kg) group exhibited a statistically significantly increase (Po0.01). It is notable that the rescue effect of 3-ODS is comparable to that of the positive control, pargyline (15 mg/kg).
2.3. Effect of 3-ODS on the number of dopaminergic neurons in the SN Tyrosine hydroxylase (TH) immunostaining of the SN was used to quantify the number of dopaminergic neurons (Fig. 4). ANOVA revealed significant differences between all groups (F¼ 74.29, Po0.0001) in the number of TH-positive cells. In the control group, the soma and fibers of dopaminergic neurons were intensely stained. In contrast, the TH-positive cells in the MPTP-treated group were only scarcely present (Po0.01). Pretreatment with 3-ODS (100 mg/kg) or pargyline (15 mg/kg) significantly increased the abundance TH-positive cells (Po0.01), suggesting 3-ODS could protect dopaminergic neurons from MPTP-induced neurotoxicity in the SN.
2.
Results
2.4.
2.1.
Effect of 3-ODS on rotarod performance
Parkinson's disease is associated with morphological atrophy and impaired synaptic function in many brain regions,
Effect of 3-ODS on spine density
In the rotarod test shown in Fig. 2, ANOVA revealed a significant differences between all groups (F¼ 60.43, Po0.0001) for the latent period. The MPTP-treated group had significantly reduced the latent period compared with
Fig. 1 – Chemical structure of 3-ODS.
Fig. 2 – 3-ODS improves motor performance in a rotarod test in the MPTP-treated mouse model of PD. Data represent the mean7SEM. (n ¼ 9). **Po0.01 compared with the MPTPtreated group; ##Po0.01 compared with the control group.
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including the hippocampus. These deficits have been attributed, at least in part, to oxidative stress. Therefore, we determined the neurotrophic effects of 3-ODS on neuronal dendrites in CA1 hippocampal neurons (Fig. 5). ANOVA revealed significant differences between all groups (F¼ 10.46, Po0.05) in the density of dendritic spines. Application of 3-
Fig. 3 – 3-ODS increases the levels of DA and its metabolite DOPAC in the striatum of the MPTP-treated mouse model of PD. Data represent the mean7SEM. (n¼ 6). **Po0.01 compared with the MPTP-treated group; ##Po0.01 compared with the control group.
ODS at a concentration of 20 μM in the culture medium for 24 h increased spine density in the secondary and tertiary branches of apical dendrites of CA1 neurons. The number of dendritic spines in the control group was 64.4374.83 spines per 100 μm of dendritic length, whereas the number of dendritic spines in 3-ODS-treated neurons was 74.7372.65 spines per 100 μm of dendritic length, resulting in a 14% increase in spine density compared with the control group (Po0.05, Fig. 5B).
2.5.
The in vitro antioxidant activities of 3-ODS
2.5.1.
Hydroxyl radical scavenging activity
The hydroxyl radical scavenging activity of 3-ODS at different concentrations is shown in Fig. 6A. The inhibitory rate of 3ODS (0.5, 1, 5, 10, 50 and 100 μM) was 471.4%, 1273.2%, 4270.5%, 5175.15%, 6970.5% and 7873.9%, respectively. The EC50 (concentration of samples required to scavenge 50% of free radicals) value of scavenging hydroxyl radical for 3-ODS was 35 μM. Therefore, in the range of 0.5–100 μM, 3-ODS scavenges hydroxyl radical in a dose-dependent manner.
Fig. 4 – 3-ODS protects on dopaminergic neurons against MPTP-induced neurotoxicity in the SN. (A, E) Control; (B, F) MPTP: 40 mg/kg; (C, G) 3-ODS: 100 mg/kg; (D, H) Pargyline: 15 mg/kg. Original magnification: A, B, C, D¼ 40 ; E, F, G, H ¼400 . Data represent the mean (% of control)7SEM of 3 mice per group and 4 sections per mouse. **Po0.01 compared with the MPTPtreated group; ##Po0.01 compared with the control group.
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Fig. 5 – 3-ODS increases dendritic spine density in hippocampal CA1 neurons. (A) Images of secondary/tertiary apical dendrites of mEGFP-expressing hippocampal CA1 neurons from control or 3-ODS treated slices. Scale bar: 2 μm. (B) Quantification of spine density, expressed per 100 μm of apical dendrites. Data represent the mean7SEM. (n¼ 3). *Po0.05 compared with the control group.
2.5.2.
Superoxide radical scavenging activity
As for the superoxide radical scavenging activity of 3-ODS (Fig. 6B), the inhibitory rate of 3-ODS (0.5, 1, 5, 10, 50 and 100 μM) was 1173.4%, 1370.3%, 1772.0%, 2473.0%, 3975.9% and 5578.3%, respectively. The EC50 value of 3-ODS was 80 μM. Again, over the range 0.5–100 μM, the scavenging activity of 3-ODS against superoxide radicals obeys a dosedependent relationship. The maximal scavenging activity of 3-ODS was observed at 100 μM.
2.5.3.
DPPH radical scavenging activity
The DPPH radical scavenging activity of 3-ODS at different concentrations is shown in Fig. 6C. The inhibitory rate of 3ODS (0.5, 1, 5, 10, 50 and 100 μM) was 274.0%, 677.3%, 1577.1%, 3276.9%, 5574.3% and 4475.9%, respectively. The EC50 value of scavenging DPPH radical for 3-ODS was 38 μM. Interestingly, the scavenging activity of 3-ODS against DPPH peaked at 50 μM and then decreased at 100 μM.
3.
Discussion
PD is a neurodegenerative disease that progresses slowly. It has been hypothesized that PD may be the result of repeated exposure to environmental toxic reagents (such as MPTP) or free radical-induced oxidative stress (Brown et al., 2005; Dexter et al., 1989; Halliwell et al., 1992; Radi et al., 2014; Reed, 2011). Therefore, it is very important to discover new drugs which reduce neurotoxic reagent-induced neuronal damage or free radical-induced oxidative stress for PD therapy. In our previous studies, we have demonstrated that the xanthone compound 3-ODS extracted from S. punicea HEMSL possesses neuroprotective effects in the H2O2 and MPPþinduced cell death in vitro. The neuroprotection imparted by
3-ODS may result from both its antioxidative and antiapoptotic activities (Zhang et al., 2010; Zhou et al., 2013). On the basis of previous in vitro results, we evaluated the neuroprotective pharmacological effects of 3-ODS in vivo for the first time. We used a MPTP-treated mouse model, which is widely used to evaluate neuroprotective compounds. This model can produce an irreversible and severe parkinsonian syndrome that replicates almost all cardinal clinical features of PD, including rigidity, tremor, impairment of balance and slowness of movement (Dujardin and Devos, 2014; Schapira and Jenner, 2011). In our present study, MPTP administration in C57BL/6 mice produced parkinsonian-like motor incoordination in the rotarod test, while pretreatment with 3-ODS was found to prevent this behavioral deficit. The effects of 3ODS were dose-dependent. The inhibitor of monoamino oxidase type B (MAO-B), pargyline, was used as a positive control in our experiments, and also improved behavioral performances (Fig. 2) in the PD mouse model, consistent with a previous study (Bazzu et al., 2013). To examine the correlation between the effects of 3-ODS on motor coordination impairment and on the neurodegenerative changes in MPTP-treated mouse model, we chose to determine the levels of DA and its metabolite DOPAC and the abundance of TH-positive neurons. DA is a major neurotransmitter released by dopaminergic neurons in the SN to the striatum, and is synthesized from tyrosine by TH, which is the rate-limiting enzyme in the DA biosynthesis. DA is metabolized into DOPAC by MAO (Nagatsua and Sawadab, 2009). It has been reported that MPTP can cause a significant decrease in the contents of DA and its metabolite in the striatum (Heikkila et al., 1984; Serra et al., 2008). Our results from the MPTP-treated group confirmed this change. Pretreatment with 3-ODS (50 and 100 mg/kg) or pargyline increased DA, and 3-ODS (25 mg/kg) or pargyline also increased the content of its metabolite DOPAC in the striatum
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Fig. 6 – Antioxidant activities of 3-ODS at various concentrations. (A) Hydroxyl radical scavenging activity of 3ODS. (B) Superoxide radical scavenging activity of 3-ODS. (C) DPPH radical scavenging activity of 3-ODS. Data represent the mean7SEM. (n ¼3).
of MPTP-treated mouse model. The DA metabolism is complicated, the DA levels may not be strictly correlated with the level of its metabolites, such as DOPAC (Guo et al., 2014). 3ODS might have differential effects on DA synthesis and degradation machineries with different effective dosages. This may be the reason why only 25 mg/kg 3-ODS was effective in rescuing DOPAC content in our experiment. The immunoreactivity for TH in the SN is one of the primary parameters reflecting the abundance of dopaminergic neurons (Meredith et al., 2008; Olanow and Kordower, 2009; Savitt et al., 2006). Thus, we used TH immunohistochemistry to confirm the anti-PD effects of 3-ODS in the MPTP-treated mice. Our data showed that the number of TH-positive neurons in the SN was significantly decreased in the MPTPtreated group, and significantly elevated by 3-ODS pretreatment. Pargyline also resulted in an increased TH immunoreactivity compared to the MPTP-treated group in our
experiment, consistent with previous reports (Bazzu et al., 2013). In summary, these results demonstrate that 3-ODS has neuroprotective effects against MPTP-induced nigrostriatal dopaminergic neurodegeneration in C57BL/6 mice, increasing the contents of DA and its metabolite DOPAC in the striatum, as well as the population of TH-positive neurons in the SN. Sufficient evidence has proven that 3-ODS has neuroprotective effects, but its beneficial actions may go beyond the dopaminergic neurons in the SN. The hippocampus is known to be involved in learning and memory processes. Although not as extensively examined as the SN and striatum in PD models, the hippocampus is implicated in many non-motor symptoms of PD (Calabresi et al., 2013). Specifically, damage to the hippocampal CA1 neurons could contribute to the impairments in working memory and recognition seen in PD (Ho et al., 2014). Hippocampal pyramidal neurons have been found to be structurally and functionally impaired in various models of parkinsonism and PD patients (Zhu et al., 2011; Wang et al., 2009; Brück et al., 2004; Camicioli et al., 2003). Therefore, drugs or compounds that increase the density of dendritic spines where most excitatory synaptic connections are formed will offer a novel potential therapeutic approach for treating neurodegenerative diseases, including PD (Smith et al., 2009; Soderstrom et al., 2010; Stephens et al., 2005). Organotypic hippocampal slice culture is a useful model system to study neuronal cell death and neuroprotection ex vivo (Holopainen, 2005). In this model system, we examined whether 3-ODS is neurotrophic towards hippocampal neurons in. In our experiments, we found that exposure to 3ODS increased the spine density in hippocampal CA1 neurons by 14% (Fig. 5), suggesting a growth-promoting effect of 3ODS. Thus, 3-ODS might be used to prevent or reverse spine loss, providing a promising therapeutic approach to alleviate cognitive impairment associated with PD. Antioxidant capacity is widely used as a parameter for evaluating medicinal bioactive compounds. Many methods, such as hydroxyl radical scavenging, superoxide anion scavenging and DPPH radical scavenging assays, were used to test the antioxidant capacity of the compound 3-ODS in vitro. The hydroxyl radical is an extremely reactive species that causes injury to surrounding biological molecules. It has been demonstrated that the reactivity of OH is related to several neurodegenerative diseases (Halliwell and Gutteridge, 1984; Halliwell et al., 1992). Therefore, removal of hydroxyl radical is the most effective defense against these diseases and has received considerable attention (Lin et al., 1995). Superoxide is a major biological source of reactive oxygen species (Alves et al., 2010). Superoxide can be decomposed to stronger oxidative species such as hydroxyl radical and singlet oxygen, which contribute to oxidative stress and are harmful to cellular components of biological systems (Zhao et al., 2006). DPPH is the most widely used stable free radical for evaluating the antioxidant capacity of compounds or plant extracts (Balakrishnan et al., 2013). This assay provides information on the reactivity of test compounds against stable free radicals. The results of our study indicate that the 3-ODS had a strong capacity of scavenging hydroxyl radical, superoxide anion and DPPH radical in a concentration-dependent manner. In particular, 3-ODS has a marked scavenging effect against the strong and toxic hydroxyl radical. The maximal inhibitory rate of 3-ODS was
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7873.9%. This suggests that 3-ODS is an effective free radical scavenger and has antioxidant activity that can protect neurons against radical-induced oxidative damage. In conclusion, the results of the present study have expanded our knowledge about the therapeutic potential of 3-ODS in the experimental models of PD in vivo and further demonstrated its suitability as a therapeutic candidate against PD or other neurodegenerative diseases. Its neuroprotective actions may involve growth-promoting effects on spine density and anti-oxidative stress (Fig.7). However, it still remains for future basic research and clinical studies to determine the potential applications of 3-ODS in PD therapy. If 3-ODS can be shown to definitively induce regeneration or prevent degeneration of neurons, it may become an important therapeutic candidate in the treatment of neurodegenerative diseases, especially PD.
4.
Experimental procedures
4.1.
Materials
S. punicea HEMSL was purchased from the Anhui Bozhou Herb Market (China) and identified by Professor Guo De-An (Department of Natural Drugs, School of Pharmaceutical Science, Peking University). A voucher specimen (SP-001) was deposited in the herbarium of the Department of Pharmacognosy, School of Pharmaceutical Sciences, Peking University. 3-ODS was extracted by Dr. Du Xin-Gang (Department of Natural Drugs, School of Pharmaceutical Science, Peking University). The purity of 3-ODS was more than 98% by high-performance liquid chromatography (HPLC) analysis. Pargyline, MPTP and DPPH were purchased from Sigma-Aldrich (USA). TH monoclonal antibody was purchased from Chemicon (U.S.A.). The hydroxyl radical kit and superoxide anion radical kit were purchased from Nanjing Jiancheng Bioengineering Institute (China). All other reagents were of analytical grade.
4.2.
Animals
Adult male C57BL/6 mice weighing 20–25 g were purchased from the Laboratory Animal Center of Peking University Health Science Center (Beijing, China). The certificate number was SCXK (JING) 2011-0012. Animals were housed under standard conditions (2272 1C) with food and water ad libitum
on a 12-h light/dark cycle. They were housed for 1 week prior to the experiments. All experiments were performed under the guidelines of the Experimental Laboratory Animal Committee of Peking University Health Science Center and were in strict accordance with the principles and guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. We minimized the number of mice used and their suffering or pain.
4.3.
MPTP-treated mouse model of PD
Mice were randomly divided into 6 groups, 9 mice per group, as follows: control group, MPTP-treated group, 3-ODS groups (25, 50 and 100 mg/kg, respectively), pargyline group (positive control, 15 mg/kg). The mice (with the exception of the control group) received four intraperitoneal injections (i.p.) of MPTP (10 mg/kg) at 1 h intervals, as described previously (Oyagi et al., 2008). The control group was injected with normal saline under the same regimen. For all groups, mice were i.p. injected twice with the respective compounds (30 min before the first MPTP treatment and 90 min after the first MPTP treatment). The control group and MPTPtreated group were injected with the same volume of normal saline. After the last administration, the mice were subjected to behavioral experiments. Three days later, the mice were decapitated, and HPLC determinations and immunohistochemical assays performed.
4.4.
The rotarod test
The mice were trained twice a day on two successive days to stay on the rotating rod (12 rpm on the first day and 18 rpm on the second day) before the test. On the third day, the mice were tested on the rotarod rod (25 rpm) three times. Each mouse was recorded over three times at 3 min intervals. Data are presented as the mean time on the rotating bar over the three test trials.
4.5.
HPLC determination of DA and DOPAC
Three days after the final MPTP treatment, 6 mice from each group were sacrificed and their brains were quickly removed and placed on ice. Their striata were dissected out and weighed. The contents of DA and its metabolite DOPAC were assayed by HPLC with electrochemical detection (ECD). HPLC analysis was performed based on previously described methods (Zhao et al., 2013).
4.6.
Fig. 7 – Schematic representation of the 3-ODS-mediated anti-PD effect.
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Immunohistochemistry of TH
Three days after the final MPTP treatment, three mice were subjected to perfusion of PBS through the aorta followed by cold 4% paraformaldehyde-PBS, under deep anesthesia with pentobarbital (50 mg/kg, i.p.). After perfusion, the brain was quickly removed and postfixed for 2 days with 4% paraformaldehyde-PBS, then transferred to 30% sucrose solution for 2 days. A series of 20 μm thick coronal sections were cut through the ventral mesencephalon using a cryostat (Leica, Nussloch, Germany). TH-immunohistochemistry in the SN sections, including microphotograph capture and data
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analysis, were performed in full accordance with the previously described protocols (Zhao et al., 2013).
4.7.
Growth-promoting effect on spine density
4.7.1.
Hippocampal slice preparation and drug treatment
Based on a previous report (Stoppini et al., 1991), organotypic hippocampal slice cultures were prepared from postnatal day 6–7 (P6-7) rats. After anesthesia, rats were decapitated and their brains removed and placed in ice-cold dissection media containing 1 mM CaCl2, 5 mM MgCl2, 10 mM glucose, 4 mM KCl, 26 mM NaHCO3 and 248 mM sucrose. Hippocampi were dissected out and coronal slices of 350 μm thickness were cut by a Mcilwain tissue chopper (Campden). Slices were then placed onto membranes (PICM0RG50, Millipore) interfacing tissue medium containing 0.0084 g/ml HEPES base MEM, 20% horse serum, 1 mM L-glutamine, 1 mM CaCl2, 2 mM MgSO4, 12.9 mM D-glucose, 5.2 mM NaHCO3, 30 mM HEPES, 0.075% ascorbic acid, and 1 μg/ml insulin (pH 7.4 and 300 mOsm). Cultured slices were incubated at 35 1C with 95% O2 and 5% CO2. After 11–13 days in vitro, CA1 pyramidal neurons were transfected with monomeric enhanced green fluorescent protein (mEGFP) by a gene-gun (Bio-Rad) (O'Brien and Lummis, 2006). One day after transfection, slices were then incubated with 3-ODS (20 μM) or vehicle (0.1% DMSO) for 24 h before imaging.
4.7.2.
Two-photon imaging and analysis
CA1 pyramidal neurons expressing mEGFP were imaged using a custom-built two-photon microscope at room temperature with a Ti: sapphire pulsed laser tuned to 920 nm (Spectra-Physics). Images were taken at high magnification (60 water-immersion objective) and further magnified using a 16 zoom. Z-stacks were acquired at 0.67-μm intervals. Apical secondary and tertiary dendrites 50–200 μm away from soma were randomly chosen for imaging (Image analysis was performed with the investigator blinded to the treatments used). Spine density was quantified manually using Image J (NIH) with the Cell Counter plug-in. Spine counting did not include protrusions in the Z-axis that could not be identified unambiguously on z-stack projection images. The total number of spines was divided by the total length of dendritic segments on which counted spines reside.
4.8.
Antioxidant activities assay
4.8.1. Hydroxyl radical and superoxide anion radical scavenging activities Scavenging activities again hydroxyl and superoxide anion radicals were measured using commercial assay kits in accordance with the manufacturer's instructions. The absorbance was read at 550 nm for both hydroxyl radical and superoxide anion radical. Scavenging activity of hydroxyl radical or superoxide anion radical (%)¼ (Acontrol Asample)/Acontrol 100%.
4.8.2.
DPPH radical scavenging activity
DPPH radical scavenging activity was measured according to the method described previously with some modifications (Blois, 1958). In a 96-microwell plate, the tested sample
(3-ODS 0.5–100 μΜ, 10 μl) or control sample (normal saline including 1% DMSO, 10 μl) were added to freshly prepared DPPH ethanol solution (65 μΜ, 190 μl). The mixture was shaken vigorously and incubated at room temperature for 30 min in the dark. The absorbance of the resulting solution was measured at 517 nm. Scavenging activity of DPPH radical (%)¼(Acontrol Asample)/ Acontrol 100%.
4.9.
Statistical analysis
Data were expressed as the mean7SEM. Statistical comparisons between groups were performed using one-way ANOVA followed by Tukey's post hoc analysis. Po0.05 was considered significant difference.
Acknowledgments This work was supported by grants from the National Instrument Development Special Program of China (No. 2013YQ03065106), Significant New Drugs Creation Five-Year Plan Special Science and Technology Major (No. 2012ZX09103201-042) and National Natural Science Funds of China (No. 81202937).
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Research report
Neuroprotective effects of 3-O-demethylswertipunicoside against MPTP-induced Parkinson's disease in vivo and its antioxidant properties in vitro Jun-Jun Zhoua,b,c, Shen-Yu Zhaia,c, Hui-Nan Zhanga,c, Yue-Hua Wangc,d, Xiao-Ping Pua,c,n a
National Key Research Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, PR China Department of Pharmacology, Dalian Medical University, Dalian 116044, PR China c Department of Molecular and Cellular Pharmacology, School of Pharmaceutical Sciences, Peking University, Beijing 100191, PR China d Beijing Key Laboratory of Drug Target Identification and Drug Screening, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, PR China b
ar t ic l e in f o
abs tra ct
Article history:
3-O-demethylswertipunicoside (3-ODS) has been reported to protect dopaminergic neurons
Accepted 30 June 2015
against neurotoxicity induced by 1-methyl-4-phenylpyridinium (MPPþ) in PC12 cells. Here, we
Available online 23 July 2015
investigate the neuroprotective effects in vivo and antioxidant activities in vitro of 3-ODS. In the 1-
Keywords:
methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP)-treated mouse model of Parkinson's disease
3-O-demethylswertipunicoside
(PD), 3-ODS dose-dependently improved motor coordination (as shown by rotarod test),
1-methyl-4-phenyl-1, 2, 3
increased the contents of dopamine (DA) and its metabolites in the striatum, and increased
6-tetrahydropyridine
the number of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra (SN). In
Parkinson's disease
addition, 3-ODS also increased the spine density in hippocampal CA1 neurons. In antioxidant
Neuroprotection
assays, 3-ODS showed a strong capacity in scavenging hydroxyl radical, superoxide anion and 1,
Growth-promoting effect
1-diphenyl-2-picrylhydrazyl (DPPH) radical in a concentration-dependent manner. Taken
Antioxidant activity
together, we conclude that 3-ODS attenuates the PD-related motor deficits mainly through its neuroprotective effects, growth-promoting effects on spine density, and its antioxidant activities. & 2015 Elsevier B.V. All rights reserved.
1.
Introduction
neurons in the substantia nigra (SN) (Hirsch et al., 1988). However, the mechanism of dopaminergic neurodegenera-
Parkinson's disease (PD) is a common neurodegenerative disease characterized by progressive loss of dopaminergic
tion is still poorly understood. The neurotoxin 1-methyl-4phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) is a mitochondrial
n Corresponding author at: National Key Research Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, PR China. Fax: þ86 10 8280 2431. E-mail address: [email protected] (X.-P. Pu).
http://dx.doi.org/10.1016/j.brainres.2015.06.051 0006-8993/& 2015 Elsevier B.V. All rights reserved.
brain research 1624 (2015) 78–85
complex I inhibitor that can cross the blood-brain barrier and be converted to its toxic metabolite 1-methyl-4phenylpyridinium (MPPþ), which selectively damages dopaminergic neurons and induces DA depletion (Anderson et al., 2006; Dauer and Przedborski, 2003). MPTP-induced parkinsonian mice provide one of the most commonly used animal models for analyzing the effects of many compounds that act on dopaminergic neurons (Patil et al., 2014; Tomac et al., 1995). It is widely accepted that oxidative stress caused by toxic free radicals plays an important role in the pathogenesis of various neurodegenerative diseases such as PD (Dexter et al., 1989; Halliwell et al., 1992; Reed, 2011). Therefore, reducing such free radicals has become a central focus of research designed to prevent or ameliorate neurodegeneration. Antioxidants from natural products or medicinal plants are being to emerge as candidates for prevention or treatment of neurodegenerative diseases. Swertia punicea HEMSL (Gentianaceae) is a traditional Chinese medicine with numerous therapeutic applications, including treatment of hepatitis, cholecystitis, and urinary tract infection (Zheng et al., 2014; Zhong et al., 2010). The xanthone compound 3-O-demethylswertipunicoside (3-ODS) is extracted from S. punicea (Du et al., 2010; Tan et al., 1992). Our previous studies have shown that 3-ODS (Fig. 1) has a neuroprotective effect against H2O2 and MPPþ-induced oxidative stress and apoptosis in PC12 cells. The mechanisms of 3ODS may involve antioxidative defense and anti-apoptotic activities (Zhang et al., 2010; Zhou et al., 2013). The aim of this study was to evaluate the neuroprotective effects and mechanisms of 3-ODS using an in vivo MPTPinduced mouse model of PD in C57 BL/6 mice and ex vivo hippocampal slice of rats. We have also investigated the possible antioxidant activities of 3-ODS using various free radical scavenging tests in vitro, such as hydroxyl radical scavenging, superoxide radical scavenging and 1, 1-diphenyl2-picryl-hydrazyl (DPPH) radical scavenging.
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the control group (Po0.01). Pretreated with higher doses of 3ODS (50 and 100 mg/kg) significantly attenuated the reduction in the latent period (both Po0.01). Pargyline (15 mg/kg), as the positive control group, also increased the latent period compared with the MPTP-treated group (Po0.01).
2.2. Effects of 3-ODS on the levels of dopamine (DA) and 3,4-dihydroxyphenyl acetic acid (DOPAC) in the striatum The effects of 3-ODS on the levels of DA and its metabolite DOPAC in the striatum are shown in Fig. 3. ANOVA revealed significant differences between all groups (F¼ 35.26, Po0.0001; F¼ 13.61, Po0.0001) for DA and DOPAC contents, respectively. DA and DOPAC contents in the MPTP-treated group were significantly lower than those in the control group (both Po0.01). After administration of medium to high doses of 3-ODS (50 and 100 mg/kg), the striatal DA contents were substantially rescued (both Po0.01). For the striatal DOPAC content, only the low-dose 3-ODS (25 mg/kg) group exhibited a statistically significantly increase (Po0.01). It is notable that the rescue effect of 3-ODS is comparable to that of the positive control, pargyline (15 mg/kg).
2.3. Effect of 3-ODS on the number of dopaminergic neurons in the SN Tyrosine hydroxylase (TH) immunostaining of the SN was used to quantify the number of dopaminergic neurons (Fig. 4). ANOVA revealed significant differences between all groups (F¼ 74.29, Po0.0001) in the number of TH-positive cells. In the control group, the soma and fibers of dopaminergic neurons were intensely stained. In contrast, the TH-positive cells in the MPTP-treated group were only scarcely present (Po0.01). Pretreatment with 3-ODS (100 mg/kg) or pargyline (15 mg/kg) significantly increased the abundance TH-positive cells (Po0.01), suggesting 3-ODS could protect dopaminergic neurons from MPTP-induced neurotoxicity in the SN.
2.
Results
2.4.
2.1.
Effect of 3-ODS on rotarod performance
Parkinson's disease is associated with morphological atrophy and impaired synaptic function in many brain regions,
Effect of 3-ODS on spine density
In the rotarod test shown in Fig. 2, ANOVA revealed a significant differences between all groups (F¼ 60.43, Po0.0001) for the latent period. The MPTP-treated group had significantly reduced the latent period compared with
Fig. 1 – Chemical structure of 3-ODS.
Fig. 2 – 3-ODS improves motor performance in a rotarod test in the MPTP-treated mouse model of PD. Data represent the mean7SEM. (n ¼ 9). **Po0.01 compared with the MPTPtreated group; ##Po0.01 compared with the control group.
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including the hippocampus. These deficits have been attributed, at least in part, to oxidative stress. Therefore, we determined the neurotrophic effects of 3-ODS on neuronal dendrites in CA1 hippocampal neurons (Fig. 5). ANOVA revealed significant differences between all groups (F¼ 10.46, Po0.05) in the density of dendritic spines. Application of 3-
Fig. 3 – 3-ODS increases the levels of DA and its metabolite DOPAC in the striatum of the MPTP-treated mouse model of PD. Data represent the mean7SEM. (n¼ 6). **Po0.01 compared with the MPTP-treated group; ##Po0.01 compared with the control group.
ODS at a concentration of 20 μM in the culture medium for 24 h increased spine density in the secondary and tertiary branches of apical dendrites of CA1 neurons. The number of dendritic spines in the control group was 64.4374.83 spines per 100 μm of dendritic length, whereas the number of dendritic spines in 3-ODS-treated neurons was 74.7372.65 spines per 100 μm of dendritic length, resulting in a 14% increase in spine density compared with the control group (Po0.05, Fig. 5B).
2.5.
The in vitro antioxidant activities of 3-ODS
2.5.1.
Hydroxyl radical scavenging activity
The hydroxyl radical scavenging activity of 3-ODS at different concentrations is shown in Fig. 6A. The inhibitory rate of 3ODS (0.5, 1, 5, 10, 50 and 100 μM) was 471.4%, 1273.2%, 4270.5%, 5175.15%, 6970.5% and 7873.9%, respectively. The EC50 (concentration of samples required to scavenge 50% of free radicals) value of scavenging hydroxyl radical for 3-ODS was 35 μM. Therefore, in the range of 0.5–100 μM, 3-ODS scavenges hydroxyl radical in a dose-dependent manner.
Fig. 4 – 3-ODS protects on dopaminergic neurons against MPTP-induced neurotoxicity in the SN. (A, E) Control; (B, F) MPTP: 40 mg/kg; (C, G) 3-ODS: 100 mg/kg; (D, H) Pargyline: 15 mg/kg. Original magnification: A, B, C, D¼ 40 ; E, F, G, H ¼400 . Data represent the mean (% of control)7SEM of 3 mice per group and 4 sections per mouse. **Po0.01 compared with the MPTPtreated group; ##Po0.01 compared with the control group.
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Fig. 5 – 3-ODS increases dendritic spine density in hippocampal CA1 neurons. (A) Images of secondary/tertiary apical dendrites of mEGFP-expressing hippocampal CA1 neurons from control or 3-ODS treated slices. Scale bar: 2 μm. (B) Quantification of spine density, expressed per 100 μm of apical dendrites. Data represent the mean7SEM. (n¼ 3). *Po0.05 compared with the control group.
2.5.2.
Superoxide radical scavenging activity
As for the superoxide radical scavenging activity of 3-ODS (Fig. 6B), the inhibitory rate of 3-ODS (0.5, 1, 5, 10, 50 and 100 μM) was 1173.4%, 1370.3%, 1772.0%, 2473.0%, 3975.9% and 5578.3%, respectively. The EC50 value of 3-ODS was 80 μM. Again, over the range 0.5–100 μM, the scavenging activity of 3-ODS against superoxide radicals obeys a dosedependent relationship. The maximal scavenging activity of 3-ODS was observed at 100 μM.
2.5.3.
DPPH radical scavenging activity
The DPPH radical scavenging activity of 3-ODS at different concentrations is shown in Fig. 6C. The inhibitory rate of 3ODS (0.5, 1, 5, 10, 50 and 100 μM) was 274.0%, 677.3%, 1577.1%, 3276.9%, 5574.3% and 4475.9%, respectively. The EC50 value of scavenging DPPH radical for 3-ODS was 38 μM. Interestingly, the scavenging activity of 3-ODS against DPPH peaked at 50 μM and then decreased at 100 μM.
3.
Discussion
PD is a neurodegenerative disease that progresses slowly. It has been hypothesized that PD may be the result of repeated exposure to environmental toxic reagents (such as MPTP) or free radical-induced oxidative stress (Brown et al., 2005; Dexter et al., 1989; Halliwell et al., 1992; Radi et al., 2014; Reed, 2011). Therefore, it is very important to discover new drugs which reduce neurotoxic reagent-induced neuronal damage or free radical-induced oxidative stress for PD therapy. In our previous studies, we have demonstrated that the xanthone compound 3-ODS extracted from S. punicea HEMSL possesses neuroprotective effects in the H2O2 and MPPþinduced cell death in vitro. The neuroprotection imparted by
3-ODS may result from both its antioxidative and antiapoptotic activities (Zhang et al., 2010; Zhou et al., 2013). On the basis of previous in vitro results, we evaluated the neuroprotective pharmacological effects of 3-ODS in vivo for the first time. We used a MPTP-treated mouse model, which is widely used to evaluate neuroprotective compounds. This model can produce an irreversible and severe parkinsonian syndrome that replicates almost all cardinal clinical features of PD, including rigidity, tremor, impairment of balance and slowness of movement (Dujardin and Devos, 2014; Schapira and Jenner, 2011). In our present study, MPTP administration in C57BL/6 mice produced parkinsonian-like motor incoordination in the rotarod test, while pretreatment with 3-ODS was found to prevent this behavioral deficit. The effects of 3ODS were dose-dependent. The inhibitor of monoamino oxidase type B (MAO-B), pargyline, was used as a positive control in our experiments, and also improved behavioral performances (Fig. 2) in the PD mouse model, consistent with a previous study (Bazzu et al., 2013). To examine the correlation between the effects of 3-ODS on motor coordination impairment and on the neurodegenerative changes in MPTP-treated mouse model, we chose to determine the levels of DA and its metabolite DOPAC and the abundance of TH-positive neurons. DA is a major neurotransmitter released by dopaminergic neurons in the SN to the striatum, and is synthesized from tyrosine by TH, which is the rate-limiting enzyme in the DA biosynthesis. DA is metabolized into DOPAC by MAO (Nagatsua and Sawadab, 2009). It has been reported that MPTP can cause a significant decrease in the contents of DA and its metabolite in the striatum (Heikkila et al., 1984; Serra et al., 2008). Our results from the MPTP-treated group confirmed this change. Pretreatment with 3-ODS (50 and 100 mg/kg) or pargyline increased DA, and 3-ODS (25 mg/kg) or pargyline also increased the content of its metabolite DOPAC in the striatum
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Fig. 6 – Antioxidant activities of 3-ODS at various concentrations. (A) Hydroxyl radical scavenging activity of 3ODS. (B) Superoxide radical scavenging activity of 3-ODS. (C) DPPH radical scavenging activity of 3-ODS. Data represent the mean7SEM. (n ¼3).
of MPTP-treated mouse model. The DA metabolism is complicated, the DA levels may not be strictly correlated with the level of its metabolites, such as DOPAC (Guo et al., 2014). 3ODS might have differential effects on DA synthesis and degradation machineries with different effective dosages. This may be the reason why only 25 mg/kg 3-ODS was effective in rescuing DOPAC content in our experiment. The immunoreactivity for TH in the SN is one of the primary parameters reflecting the abundance of dopaminergic neurons (Meredith et al., 2008; Olanow and Kordower, 2009; Savitt et al., 2006). Thus, we used TH immunohistochemistry to confirm the anti-PD effects of 3-ODS in the MPTP-treated mice. Our data showed that the number of TH-positive neurons in the SN was significantly decreased in the MPTPtreated group, and significantly elevated by 3-ODS pretreatment. Pargyline also resulted in an increased TH immunoreactivity compared to the MPTP-treated group in our
experiment, consistent with previous reports (Bazzu et al., 2013). In summary, these results demonstrate that 3-ODS has neuroprotective effects against MPTP-induced nigrostriatal dopaminergic neurodegeneration in C57BL/6 mice, increasing the contents of DA and its metabolite DOPAC in the striatum, as well as the population of TH-positive neurons in the SN. Sufficient evidence has proven that 3-ODS has neuroprotective effects, but its beneficial actions may go beyond the dopaminergic neurons in the SN. The hippocampus is known to be involved in learning and memory processes. Although not as extensively examined as the SN and striatum in PD models, the hippocampus is implicated in many non-motor symptoms of PD (Calabresi et al., 2013). Specifically, damage to the hippocampal CA1 neurons could contribute to the impairments in working memory and recognition seen in PD (Ho et al., 2014). Hippocampal pyramidal neurons have been found to be structurally and functionally impaired in various models of parkinsonism and PD patients (Zhu et al., 2011; Wang et al., 2009; Brück et al., 2004; Camicioli et al., 2003). Therefore, drugs or compounds that increase the density of dendritic spines where most excitatory synaptic connections are formed will offer a novel potential therapeutic approach for treating neurodegenerative diseases, including PD (Smith et al., 2009; Soderstrom et al., 2010; Stephens et al., 2005). Organotypic hippocampal slice culture is a useful model system to study neuronal cell death and neuroprotection ex vivo (Holopainen, 2005). In this model system, we examined whether 3-ODS is neurotrophic towards hippocampal neurons in. In our experiments, we found that exposure to 3ODS increased the spine density in hippocampal CA1 neurons by 14% (Fig. 5), suggesting a growth-promoting effect of 3ODS. Thus, 3-ODS might be used to prevent or reverse spine loss, providing a promising therapeutic approach to alleviate cognitive impairment associated with PD. Antioxidant capacity is widely used as a parameter for evaluating medicinal bioactive compounds. Many methods, such as hydroxyl radical scavenging, superoxide anion scavenging and DPPH radical scavenging assays, were used to test the antioxidant capacity of the compound 3-ODS in vitro. The hydroxyl radical is an extremely reactive species that causes injury to surrounding biological molecules. It has been demonstrated that the reactivity of OH is related to several neurodegenerative diseases (Halliwell and Gutteridge, 1984; Halliwell et al., 1992). Therefore, removal of hydroxyl radical is the most effective defense against these diseases and has received considerable attention (Lin et al., 1995). Superoxide is a major biological source of reactive oxygen species (Alves et al., 2010). Superoxide can be decomposed to stronger oxidative species such as hydroxyl radical and singlet oxygen, which contribute to oxidative stress and are harmful to cellular components of biological systems (Zhao et al., 2006). DPPH is the most widely used stable free radical for evaluating the antioxidant capacity of compounds or plant extracts (Balakrishnan et al., 2013). This assay provides information on the reactivity of test compounds against stable free radicals. The results of our study indicate that the 3-ODS had a strong capacity of scavenging hydroxyl radical, superoxide anion and DPPH radical in a concentration-dependent manner. In particular, 3-ODS has a marked scavenging effect against the strong and toxic hydroxyl radical. The maximal inhibitory rate of 3-ODS was
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7873.9%. This suggests that 3-ODS is an effective free radical scavenger and has antioxidant activity that can protect neurons against radical-induced oxidative damage. In conclusion, the results of the present study have expanded our knowledge about the therapeutic potential of 3-ODS in the experimental models of PD in vivo and further demonstrated its suitability as a therapeutic candidate against PD or other neurodegenerative diseases. Its neuroprotective actions may involve growth-promoting effects on spine density and anti-oxidative stress (Fig.7). However, it still remains for future basic research and clinical studies to determine the potential applications of 3-ODS in PD therapy. If 3-ODS can be shown to definitively induce regeneration or prevent degeneration of neurons, it may become an important therapeutic candidate in the treatment of neurodegenerative diseases, especially PD.
4.
Experimental procedures
4.1.
Materials
S. punicea HEMSL was purchased from the Anhui Bozhou Herb Market (China) and identified by Professor Guo De-An (Department of Natural Drugs, School of Pharmaceutical Science, Peking University). A voucher specimen (SP-001) was deposited in the herbarium of the Department of Pharmacognosy, School of Pharmaceutical Sciences, Peking University. 3-ODS was extracted by Dr. Du Xin-Gang (Department of Natural Drugs, School of Pharmaceutical Science, Peking University). The purity of 3-ODS was more than 98% by high-performance liquid chromatography (HPLC) analysis. Pargyline, MPTP and DPPH were purchased from Sigma-Aldrich (USA). TH monoclonal antibody was purchased from Chemicon (U.S.A.). The hydroxyl radical kit and superoxide anion radical kit were purchased from Nanjing Jiancheng Bioengineering Institute (China). All other reagents were of analytical grade.
4.2.
Animals
Adult male C57BL/6 mice weighing 20–25 g were purchased from the Laboratory Animal Center of Peking University Health Science Center (Beijing, China). The certificate number was SCXK (JING) 2011-0012. Animals were housed under standard conditions (2272 1C) with food and water ad libitum
on a 12-h light/dark cycle. They were housed for 1 week prior to the experiments. All experiments were performed under the guidelines of the Experimental Laboratory Animal Committee of Peking University Health Science Center and were in strict accordance with the principles and guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. We minimized the number of mice used and their suffering or pain.
4.3.
MPTP-treated mouse model of PD
Mice were randomly divided into 6 groups, 9 mice per group, as follows: control group, MPTP-treated group, 3-ODS groups (25, 50 and 100 mg/kg, respectively), pargyline group (positive control, 15 mg/kg). The mice (with the exception of the control group) received four intraperitoneal injections (i.p.) of MPTP (10 mg/kg) at 1 h intervals, as described previously (Oyagi et al., 2008). The control group was injected with normal saline under the same regimen. For all groups, mice were i.p. injected twice with the respective compounds (30 min before the first MPTP treatment and 90 min after the first MPTP treatment). The control group and MPTPtreated group were injected with the same volume of normal saline. After the last administration, the mice were subjected to behavioral experiments. Three days later, the mice were decapitated, and HPLC determinations and immunohistochemical assays performed.
4.4.
The rotarod test
The mice were trained twice a day on two successive days to stay on the rotating rod (12 rpm on the first day and 18 rpm on the second day) before the test. On the third day, the mice were tested on the rotarod rod (25 rpm) three times. Each mouse was recorded over three times at 3 min intervals. Data are presented as the mean time on the rotating bar over the three test trials.
4.5.
HPLC determination of DA and DOPAC
Three days after the final MPTP treatment, 6 mice from each group were sacrificed and their brains were quickly removed and placed on ice. Their striata were dissected out and weighed. The contents of DA and its metabolite DOPAC were assayed by HPLC with electrochemical detection (ECD). HPLC analysis was performed based on previously described methods (Zhao et al., 2013).
4.6.
Fig. 7 – Schematic representation of the 3-ODS-mediated anti-PD effect.
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Immunohistochemistry of TH
Three days after the final MPTP treatment, three mice were subjected to perfusion of PBS through the aorta followed by cold 4% paraformaldehyde-PBS, under deep anesthesia with pentobarbital (50 mg/kg, i.p.). After perfusion, the brain was quickly removed and postfixed for 2 days with 4% paraformaldehyde-PBS, then transferred to 30% sucrose solution for 2 days. A series of 20 μm thick coronal sections were cut through the ventral mesencephalon using a cryostat (Leica, Nussloch, Germany). TH-immunohistochemistry in the SN sections, including microphotograph capture and data
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analysis, were performed in full accordance with the previously described protocols (Zhao et al., 2013).
4.7.
Growth-promoting effect on spine density
4.7.1.
Hippocampal slice preparation and drug treatment
Based on a previous report (Stoppini et al., 1991), organotypic hippocampal slice cultures were prepared from postnatal day 6–7 (P6-7) rats. After anesthesia, rats were decapitated and their brains removed and placed in ice-cold dissection media containing 1 mM CaCl2, 5 mM MgCl2, 10 mM glucose, 4 mM KCl, 26 mM NaHCO3 and 248 mM sucrose. Hippocampi were dissected out and coronal slices of 350 μm thickness were cut by a Mcilwain tissue chopper (Campden). Slices were then placed onto membranes (PICM0RG50, Millipore) interfacing tissue medium containing 0.0084 g/ml HEPES base MEM, 20% horse serum, 1 mM L-glutamine, 1 mM CaCl2, 2 mM MgSO4, 12.9 mM D-glucose, 5.2 mM NaHCO3, 30 mM HEPES, 0.075% ascorbic acid, and 1 μg/ml insulin (pH 7.4 and 300 mOsm). Cultured slices were incubated at 35 1C with 95% O2 and 5% CO2. After 11–13 days in vitro, CA1 pyramidal neurons were transfected with monomeric enhanced green fluorescent protein (mEGFP) by a gene-gun (Bio-Rad) (O'Brien and Lummis, 2006). One day after transfection, slices were then incubated with 3-ODS (20 μM) or vehicle (0.1% DMSO) for 24 h before imaging.
4.7.2.
Two-photon imaging and analysis
CA1 pyramidal neurons expressing mEGFP were imaged using a custom-built two-photon microscope at room temperature with a Ti: sapphire pulsed laser tuned to 920 nm (Spectra-Physics). Images were taken at high magnification (60 water-immersion objective) and further magnified using a 16 zoom. Z-stacks were acquired at 0.67-μm intervals. Apical secondary and tertiary dendrites 50–200 μm away from soma were randomly chosen for imaging (Image analysis was performed with the investigator blinded to the treatments used). Spine density was quantified manually using Image J (NIH) with the Cell Counter plug-in. Spine counting did not include protrusions in the Z-axis that could not be identified unambiguously on z-stack projection images. The total number of spines was divided by the total length of dendritic segments on which counted spines reside.
4.8.
Antioxidant activities assay
4.8.1. Hydroxyl radical and superoxide anion radical scavenging activities Scavenging activities again hydroxyl and superoxide anion radicals were measured using commercial assay kits in accordance with the manufacturer's instructions. The absorbance was read at 550 nm for both hydroxyl radical and superoxide anion radical. Scavenging activity of hydroxyl radical or superoxide anion radical (%)¼ (Acontrol Asample)/Acontrol 100%.
4.8.2.
DPPH radical scavenging activity
DPPH radical scavenging activity was measured according to the method described previously with some modifications (Blois, 1958). In a 96-microwell plate, the tested sample
(3-ODS 0.5–100 μΜ, 10 μl) or control sample (normal saline including 1% DMSO, 10 μl) were added to freshly prepared DPPH ethanol solution (65 μΜ, 190 μl). The mixture was shaken vigorously and incubated at room temperature for 30 min in the dark. The absorbance of the resulting solution was measured at 517 nm. Scavenging activity of DPPH radical (%)¼(Acontrol Asample)/ Acontrol 100%.
4.9.
Statistical analysis
Data were expressed as the mean7SEM. Statistical comparisons between groups were performed using one-way ANOVA followed by Tukey's post hoc analysis. Po0.05 was considered significant difference.
Acknowledgments This work was supported by grants from the National Instrument Development Special Program of China (No. 2013YQ03065106), Significant New Drugs Creation Five-Year Plan Special Science and Technology Major (No. 2012ZX09103201-042) and National Natural Science Funds of China (No. 81202937).
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