Abstract

Introduction

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by major behavioral symptoms, which include tremor, akinesia, bradykinesia, and stiffness (Jankovic, 2008Jankovic J (2008) Parkinson’s disease: Clinical features and diagnosis. J Neurol Neurosurg Psychiatry 79:68–376.). PD results from the death of dopaminergic neurons in the substantia nigra (SN; (Goto et al., 1989Goto S, Hirano A and Matsumoto S (1989) Subdivisional involvement of nigrostriatal loop in idiopathic Parkinson’s disease and striatonigral degeneration. Ann Neurol 26:766–770.; Chauhan et al., 2001Chauhan NB, Siegel GJ and Lee JM (2001) Depletion of glial cell line-derived neurotrophic factor in substantia nigra neurons of Parkinson’s disease brain. J Chem Neuroanat 21:277–288.; Kim et al., 2003Kim SJ, Sung JY, Um JW, Hattori N, Mizuno Y, Tanaka K, Paik SR, Kim J and Chung KC (2003) Parkin cleaves intracellular alpha-synuclein inclusions via the activation of calpain. J Biol Chem 278:41890–41899.; Fernandez-Espejo 2004Fernandez-Espejo E (2004) Pathogenesis of Parkinson’s disease: Prospects of neuroprotective and restorative therapies. Mol Neurobiol 29:15–30.), which plays an important role in several brain functions, particularly, motor planning, eye movement, learning, reward-seeking and addiction. Not surprisingly, gross movement dysfunction caused by damage to this area is a symptom of PD.

Using a 6-hydroxydopamine (6-OHDA)-induced Parkinsonism model, a previous study found that acupuncture stimulation at acupoints GB34 and LR3 results in the enhanced survival of dopaminergic neurons in the SN and a ∼87.7% improvement in motor dysfunction (Park et al., 2003Park HJ, Lim S, Joo WS, Yin CS, Lee HS, Lee HJ, Seo JC, Leem K, Son YS, Kim YJ, et al. (2003) Acupuncture prevents 6-hydroxydopamine-induced neuronal death in the nigrostriatal dopaminergic system in the rat Parkinson’s disease model. Exp Neurol 180:93–98.). Similar finding were found following the administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), where the acupuncture-induced enhancement of synaptic dopamine availability may play a critical role in the observed motor function improvement (Kim et al., 2011Kim SN, Doo AR, Park JY, Bae H, Chae Y, Shim I, Lee H, Moon W and Park HJ (2011) Acupuncture enhances the synaptic dopamine availability to improve motor function in a mouse model of Parkinson’s disease. PLoS One 6:e27566.). Acupuncture stimulation at the same acupoints also attenuates the decrease in tyrosine hydroxylase (TH) immunoreactivity (IR) and generates neuroprotective effects in the SN in an MPTP-induced mouse model of Parkinsonism (Kang et al., 2007Kang JM, Park HJ, Choi YG, Choe IH, Park JH, Kim YS and Lim S (2007) Acupuncture inhibits microglial activation and inflammatory events in the mptp-induced mouse model. Brain Res 1131:211–219.). The mechanisms underlying the neuroprotective effects of acupuncture on the SN are thought to be in part related to decreased microglial activation and reduced inflammatory responses (Kang et al., 2007Kang JM, Park HJ, Choi YG, Choe IH, Park JH, Kim YS and Lim S (2007) Acupuncture inhibits microglial activation and inflammatory events in the mptp-induced mouse model. Brain Res 1131:211–219.); these, in turn, inhibit ferric iron and ferritin heavy chain deposition (Choi et al., 2009Choi YG, Park JH and Lim S (2009) Acupuncture inhibits ferric iron deposition and ferritin-heavy chain reduction in an mptp-induced Parkinsonism model. Neurosci Lett 450:92–96.).

Several genes that are affected by MPTP and stimulation at acupoints may be responsible for the inhibitory effects of acupuncture on MPTP-induced degeneration in striatal regions (Choi et al., 2011bChoi YG, Yeo S, Hong YM and Lim S (2011b) Neuroprotective changes of striatal degeneration-related gene expression by acupuncture in an mptp mouse model of Parkinsonism: Microarray analysis. Cell Mol Neurobiol 31:377–391.). Additionally, gene expression in thalamic regions damaged by MPTP intoxication may be affected, at least in part, by acupuncture at specific acupoints (Yeo et al., 2013Yeo S, Choi YG, Hong YM and Lim S (2013) Neuroprotective changes of thalamic degeneration-related gene expression by acupuncture in an MTPT mouse model of Parkinsonism: Microarray analysis. Gene 515:329–338.). Changes in gene expression profiles in the SN following acupuncture stimulation were identified in an acute Parkinsonism model over three days (Hong et al., 2010Hong MS, Park HK, Yang JS, Park HJ, Kim ST, Kim SN, Park JY, Song JY, Jo DJ, Park SW, et al. (2010) Gene expression profile of acupuncture treatment in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinson’s disease model. Neurol Res 32(Suppl 1):74–78.); however, no such changes have been demonstrated in a chronic Parkinsonism animal model. Because PD is a chronic neurodegenerative disorder, changes in gene expression in the SN in a chronic Parkinsonism animal model should be investigated to identify the neuroprotective mechanisms underlying acupuncture treatment.

Thus, we investigated changes in gene expression profiles in the SN region in a relatively chronic Parkinsonism model and identified the genes related to the inhibitory effect of acupuncture on alterations in gene expression induced by MPTP intoxication.

Materials and Methods

MPTP model of Parkinsonism

Six-week-old male inbred C57BL/6 mice (20–22 g; Samtaco, Korea) were divided into the following four groups: control (CTL); MPTP-treatment only (MPTP); MPTP and acupuncture treatment at GB34 and LR3 (MPTP-A); and MPTP and acupuncture treatment at nonacupoints (MPTP-NA). Mice in the control group (n = 9) were injected intraperitoneally with saline 0.9% (100 μL) once daily for four weeks, while mice in the MPTP group (n = 9) were injected intraperitoneally with MPTP-HCl (20 mg/kg of free base) in saline 0.9% (100 μL) at 24-h intervals over four weeks to produce a sustained chronic model of Parkinsonism (Bezard et al., 1997Bezard E, Dovero S, Bioulac B and Gross C (1997) Effects of different schedules of MPTP administration on dopaminergic neurodegeneration in mice. Exp Neurol 148:288–292.; Kuhn et al., 2003Kuhn K, Wellen J, Link N, Maskri L, Lubbert H and Stichel CC (2003) The mouse MPTP model: Gene expression changes in dopaminergic neurons. Eur J Neurosci 17:1–12.; Choi et al., 2011aChoi YG, Yeo S, Hong YM, Kim SH and Lim S (2011a) Changes of gene expression profiles in the cervical spinal cord by acupuncture in an MPTP-intoxicated mouse model: Microarray analysis. Gene 481:7–16.; Yeo et al., 2013Yeo S, Choi YG, Hong YM and Lim S (2013) Neuroprotective changes of thalamic degeneration-related gene expression by acupuncture in an MTPT mouse model of Parkinsonism: Microarray analysis. Gene 515:329–338.). On the day following the final MPTP treatment, mice were anesthetized using 16.5% urethane and perfused transcardially with cold 0.05 M sodium-phosphate buffer to enable immunohisto-chemical evaluation. The Kyung Hee University Animal Experimentation Committee approved all animal protocols used in this study. Reagents used but not mentioned were purchased from Sigma (USA).

Acupuncture administration

Acupuncture was performed manually 2 h after the first MPTP injection and at 48-h intervals thereafter for a total of 14 sessions. The acupuncture procedure (acupoints: GB34 [Yanglingquan] and LR3 [Taichong]; non-acupoints: both sides of the hips) was performed as reported previously (Park et al., 2003Park HJ, Lim S, Joo WS, Yin CS, Lee HS, Lee HJ, Seo JC, Leem K, Son YS, Kim YJ, et al. (2003) Acupuncture prevents 6-hydroxydopamine-induced neuronal death in the nigrostriatal dopaminergic system in the rat Parkinson’s disease model. Exp Neurol 180:93–98.; Kang et al., 2007Kang JM, Park HJ, Choi YG, Choe IH, Park JH, Kim YS and Lim S (2007) Acupuncture inhibits microglial activation and inflammatory events in the mptp-induced mouse model. Brain Res 1131:211–219.; Choi et al., 2011bChoi YG, Yeo S, Hong YM and Lim S (2011b) Neuroprotective changes of striatal degeneration-related gene expression by acupuncture in an mptp mouse model of Parkinsonism: Microarray analysis. Cell Mol Neurobiol 31:377–391.). Briefly, mice in the acupoints group were immobilized by hand 2 h following MPTP administration, and the acupuncture needles were inserted bilaterally to depths of 1 mm at acupoint LR3 and 3 mm at acupoint GB34 before being turned at a rate of two spins per second for 15 s, as reported previously (Kang et al., 2007Kang JM, Park HJ, Choi YG, Choe IH, Park JH, Kim YS and Lim S (2007) Acupuncture inhibits microglial activation and inflammatory events in the mptp-induced mouse model. Brain Res 1131:211–219.). In the non-acupoints group, the needles were inserted to a depth of 3 mm on both sides of the hips, and a procedure identical to that described above was performed.

Immunohistochemistry

After four weeks, the brains were removed, postfixed in 0.05 M sodium-phosphate buffer containing 4% paraformaldehyde for 12 h at 4 °C, rinsed with 0.05 M sodium-phosphate buffer, dehydrated with sucrose for 12 h at 4°C, and then cryosectioned. Coronal sections of the brains (30-μm thickness) were cut using a cryomicrotome. Immunohistochemical analyses were carried out using an ABC kit and a Mouse on Mouse (M.O.M) immunodetection kit (Vector Laboratories, CA) using a modification of the avidin-biotin-peroxidase method. Briefly, sections encompassing the entire striatal and SN regions were incubated in phosphate-buffered saline (PBS; pH 7.4) with 3% H₂O₂, exposed to 3% bovine serum albumin (BSA) and 0.3% Triton X-100 in PBS for 1 h, and then treated with an avidinbiotin blocking kit (Vector Laboratories). When using the mouse anti-TH antibody, sections were treated with an M.O.M. mouse Ig-blocking reagent (Vector Laboratories) at room temperature for 1 h prior to incubation with the primary antibody. Thereafter, each section was stained overnight at 4 °C with a mouse anti-TH antibody (1:1,500; Santa Cruz Biotechnology, USA) to identify dopaminergic neurons in the striatal and SN regions. The sections were then sequentially treated with a biotinylated anti-mouse IgG followed by an avidin-biotin-peroxidase complex and developed using a diaminobenzidine-hydrogen peroxide solution (0.003% 3,3-diaminobenzidine and 0.03% hydrogen peroxide in 0.05 M Tris, pH 7.0).

Western blotting

The bilateral striatal and SN regions were homogenized in 20 mM HEPES-KOH buffer (pH 7.5) with 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, and a protease inhibitor cocktail. After centrifugation at 15,000 × g for 10 min, soluble supernatant samples of equal protein concentration (30 μg total protein) were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad; USA). The membranes were blocked with 5% skim milk in 0.1% Tris-buffered saline (TBS; 20 mM Tris-HCl [pH 7.5] and 150 mM NaCl containing 0.1% Tween-20; TBST) at room temperature for 1 h and then incubated with mouse anti-TH (1:2,000) and mouse anti-actin (1:5,000, Santa Cruz Bio-technology) antibodies. After being washed with 0.1% TBST, the membranes were incubated with an anti-mouse IgG-peroxidase antibody (1:2,000, Bio-Rad), and the antigen-antibody complexes were visualized using the Pierce ECL western blotting substrate (Thermo Scientific, USA).

RNA extraction and microarray analysis

Total RNA was extracted from the bilateral SN tissue of each group (n = 2; both groups) using an RNeasy Plus Mini kit (QIAGEN, USA) according to the manufacturer’s instructions. Isolated RNA quality was estimated and quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, USA). An aliquot (300 ng) of total RNA was subjected to an Affymetrix GeneChip^® Mouse Gene 1.0 ST Array (genome-wide expression profiling chip; 28,853 genes of 35,557 probes; Affymetrix, USA) according to the GeneChip Whole Transcript (WT) Sense Target Labeling Assay manual, as reported previously (Choi et al., 2011aChoi YG, Yeo S, Hong YM, Kim SH and Lim S (2011a) Changes of gene expression profiles in the cervical spinal cord by acupuncture in an MPTP-intoxicated mouse model: Microarray analysis. Gene 481:7–16.; Hwang et al., 2009Hwang SH, Choi YG, Jeong MY, Hong YM, Lee JH and Lim S (2009) Microarray analysis of gene expression profile by treatment of cinnamomi ramulus in lipopolysaccharide-stimulated BV-2 cells. Gene 443:83–90.; Lin et al., 2010Lin HQ, Choi R, Chan KL, Ip D, Tsim KW and Wan DC (2010) Differential gene expression profiling on the muscle of acetylcholinesterase knockout mice: A preliminary analysis. Chem Biol Interact 187:120–123.). Briefly, the procedures were carried out as follows: mixing of T7-(N)6 primer and poly-A RNA controls with 300 ng of total RNA isolated from the SN of each group; synthesis of first-cycle, first-strand complementary DNA (cDNA) and then second-strand cDNA; synthesis of first-cycle cRNA and cleanup of cRNA; synthesis of second-cycle, single-strand cDNA (ss cDNA) and cleanup of ss cDNA; fragmentation of ss cDNA and labeling of the fragmented ss cDNA; hybridization of labeled ss cDNA to the GeneChip; and finally, the staining, washing, and scanning of the GeneChip using a Fluidics 450 station and the GeneChip Operating Software (GCOS, Affymetrix).

Microarray data analysis

The usefulness of the signal intensities was first evaluated by visual examination of the scanned images. Quality control of the scanned data was then conducted by confirming the order of the signal intensities of poly-A controls and hybridization controls using the Expression Console software (Affymetrix). Microarray data were analyzed using GenPlex ver. 3.0 (ISTECH, Korea, (An et al., 2009An JH, Lee SY, Jeon JY, Cho KG, Kim SU and Lee MA (2009) Identification of gliotropic factors that induce human stem cell migration to malignant tumor. J Proteome Res 8:2873–2881.; Choi et al., 2011aChoi YG, Yeo S, Hong YM, Kim SH and Lim S (2011a) Changes of gene expression profiles in the cervical spinal cord by acupuncture in an MPTP-intoxicated mouse model: Microarray analysis. Gene 481:7–16.; Hwang et al., 2009Hwang SH, Choi YG, Jeong MY, Hong YM, Lee JH and Lim S (2009) Microarray analysis of gene expression profile by treatment of cinnamomi ramulus in lipopolysaccharide-stimulated BV-2 cells. Gene 443:83–90.; Yeo et al., 2013Yeo S, Choi YG, Hong YM and Lim S (2013) Neuroprotective changes of thalamic degeneration-related gene expression by acupuncture in an MTPT mouse model of Parkinsonism: Microarray analysis. Gene 515:329–338.). A total of eight CEL files (two CEL files generated from each group x four experimental groups) were uploaded and normalized under the following conditions: perfect match (PM)-only as a PM intensity adjustment; the Robust Multichip Analysis (RMA) quantification method as a probe set summarization algorithm for log transformation with base 2 (log2); and the quantile normalization method for evaluation of preliminary data quality in the preprocessing module, which functions as a data quality control, using the Expression Console software (Affymetrix). The mean signal intensities of the 28,853 genes were obtained from two chips from each group. Following normalization, the differentially expressed genes (DEGs) in the GeneChip that satisfied the conditions of the fold change cutoff (1.3) as well as the Student’s t-test significance criterion (p < 0.05) were identified using the DEG-finding module; i.e., log1.3/log2 = 0.379 < average log2 fold-change value (X) for the identification of up-regulated genes and - log1.3/log2 = −0.379 > average log2 fold-change value (X) for the identification of down-regulated genes. A pathway analysis of all metabolic and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways was performed using the pathway module, as described previously (Hwang et al., 2009Hwang SH, Choi YG, Jeong MY, Hong YM, Lee JH and Lim S (2009) Microarray analysis of gene expression profile by treatment of cinnamomi ramulus in lipopolysaccharide-stimulated BV-2 cells. Gene 443:83–90.; Choi et al., 2011aChoi YG, Yeo S, Hong YM, Kim SH and Lim S (2011a) Changes of gene expression profiles in the cervical spinal cord by acupuncture in an MPTP-intoxicated mouse model: Microarray analysis. Gene 481:7–16.; Yeo et al., 2013Yeo S, Choi YG, Hong YM and Lim S (2013) Neuroprotective changes of thalamic degeneration-related gene expression by acupuncture in an MTPT mouse model of Parkinsonism: Microarray analysis. Gene 515:329–338.). A National Center for Biotechnology Information Gene Expression Omnibus (NCBI GEO) accession number was assigned to the raw microarray data generated in this study.

Real time RT-PCR

For real-time reverse transcription polymerase chain reaction (RT-PCR) analyses, the total RNA (500 ng) of the samples (CTL, MPTP, and MPTP-A) was subjected to the reaction using a SuperScript First-strand Synthesis Kit (Invitrogen Life Technologies, USA) according to the manufacturer’s instructions. The advanced relative expression levels of representative DEGs were monitored with a LightCycler 480 II Real Time PCR Instrument and the LightCycler 480 software ver. 1.5.0.39 (Roche Diagnostics; Germany) using a LightCycler 480 SYBR Green I Master (Roche Diagnostics). Mean crossing point (CP) values were obtained and the expression levels of the target genes in the various groups were then compared to those of the reference gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), using an advanced relative quantification method. The ratios of the concentrations of target genes to that of the reference gene were obtained; the primer sequences for each gene are shown in Table 1. The specificity of each primer set was confirmed by determining the melting temperature and size of each product by gel electrophoresis.

Statistical analysis

Statistical analyses were performed using the statistical package for the social sciences (SPSS) software, ver. 18. Values are means ± SE of three independent determinations.

Results and Discussion

Chronic MPTP-induced Parkinsonism mouse model and the preventive effects of acupuncture

Because TH expression is significantly attenuated in the brains of PD patients (Pardridge, 2005Pardridge WM (2005) Tyrosine hydroxylase replacement in experimental Parkinson’s disease with transvascular gene therapy. NeuroRx 2:129–138.) and in Parkinsonism animal models (Park et al., 2003Park HJ, Lim S, Joo WS, Yin CS, Lee HS, Lee HJ, Seo JC, Leem K, Son YS, Kim YJ, et al. (2003) Acupuncture prevents 6-hydroxydopamine-induced neuronal death in the nigrostriatal dopaminergic system in the rat Parkinson’s disease model. Exp Neurol 180:93–98.; Kang et al., 2007Kang JM, Park HJ, Choi YG, Choe IH, Park JH, Kim YS and Lim S (2007) Acupuncture inhibits microglial activation and inflammatory events in the mptp-induced mouse model. Brain Res 1131:211–219.; Choi et al., 2009Choi YG, Park JH and Lim S (2009) Acupuncture inhibits ferric iron deposition and ferritin-heavy chain reduction in an mptp-induced Parkinsonism model. Neurosci Lett 450:92–96.), we evaluated TH levels to confirm the establishment of a chronic MPTP-induced Parkinsonism mouse model and the mediating effects of acupuncture at GB34 and LR4. In the SN pars compacta (SNpc) and striatal regions, TH levels were noticeably reduced in the MPTP group (MPTP) relative to the control group (CTL; Figures 1a, b, e, f, i, and j). Acupuncture stimulation at GB34 and LR3 (MPTP-A) attenuated the decrease in TH in both areas (Figures 1c, g, and k), but acupuncture at nonacupoints (MPTP-NA) did not similarly influence TH (Figures 1d, h, and l). Immunohistochemical analyses using Western blots confirmed that the decrease in TH levels in the striatal and SNpc regions induced by MPTP intoxication was significantly inhibited by acupuncture at acupoints (MPTP-A) but not at non-acupoints (MPTP-NA; Figure 2). These results confirm that acupuncture stimulation suppresses the pathological change in TH levels induced by MPTP intoxication in the striatal and SNpc regions, of the mice in our study.

Changes in gene expression in the SN region following chronic MPTP intoxication

As shown in the box plot graph (Figure S1) normalization through the preprocessing module was successful. Of the 28,835 genes represented in the oligonucleotide array, genes were selected and evaluated if they displayed log2-transformed mean signal intensities greater than 0.379 (1.3 fold change) and p < 0.05 by Student’s t-test between the control and MPTP groups. Compared to the control, 244 up-regulated (Table S1) and 255 down-regulated (Table S2) DEGs were identified in the SN region following chronic MPTP intoxication.

Changes in gene expression in the SN region following acupuncture

Of the 28,853 genes represented in the oligonucleotide array, those with log2-transformed mean signal intensities greater than 0.379 (1.3-fold change) and a p < 0.05 by Student’s t-test between the MPTP-A and MPTP groups or the MPTP-NA and MPTP groups were selected and evaluated. Compared to the MPTP group, genes in the SN region were regarded as DEGs following acupuncture stimulation at acupoints and non-acupoints, respectively.

Up-down-regulated genes following MPTP intoxication and acupuncture

Compared to the control, 22 (10 annotated genes) of the 244 probes were up-regulated in the SN region following MPTP exposure and exclusively down-regulated by acupuncture at acupoints but not non-acupoints (“Up-down”; Table 2).

Cadherin 1 (Cdh1) is a classic member of the cadherin superfamily that is thought to contribute to the control of synapse formation and synaptic transmission and plasticity (Angst et al., 2001Angst BD, Marcozzi C and Magee AI (2001) The cadherin super-family: Diversity in form and function. J Cell Sci 114:629–641.). Cdh1 signaling is an important component of the regulation of GABAergic synapses in brain neurons (Fiederling et al., 2011Fiederling A, Ewert R, Andreyeva A, Jungling K and Gottmann K (2011) E-cadherin is required at gabaergic synapses in cultured cortical neurons. Neurosci Lett 501:167–172.); indeed, elevated GABA levels have been detected in PD (Emir et al., 2012Emir UE, Tuite PJ and Oz G (2012) Elevated pontine and putamenal gaba levels in mild-moderate Parkinson disease detected by 7 tesla proton MRS. PLoS One 7:e30918.). In the current study, Cdh1 was up-regulated in the MPTP group compared to controls. However, acupuncture stimulation at GB34 and LR3 maintained Cdh1 expression similar to levels in control animals, suggesting that the down-regulation of Cdh1 expression by acupuncture may have a neuroprotective effect.

Solute carrier family 6 (neurotransmitter transporter, GABA), member 13 (Slc6a13) is a sodium- and chloride-dependent member of solute carrier family 6 (SLC6) that mediates the rapid removal of GABA and maintains low extracellular levels of this neurotransmitter (Chen et al., 2004Chen NH, Reith ME and Quick MW (2004) Synaptic uptake and beyond: The sodium- and chloride-dependent neurotransmitter transporter family SLC6. Pflügers Arch 447:519–531.; Kanner, 2006Kanner BI (2006) Structure and function of sodium-coupled GABA and glutamate transporters. J Membr Biol 213:89–100.). Evidence suggests that alteration of GABA transporters is linked to PD (Wang et al., 2007Wang H, Katz J, Dagostino P and Soghomonian JJ (2007) Unilateral 6-hydroxydopamine lesion of dopamine neurons and subchronic l-dopa administration in the adult rat alters the expression of the vesicular gaba transporter in different subsets of striatal neurons and in the substantia nigra, pars reticulata. Neuroscience 145:727–737.). Accordingly, the overstimulation of GABA_A receptors can potentiate neuronal cell damage; GABA transport blockers are neuroprotective in these situations (Zeevalk and Nicklas, 1996Zeevalk GD and Nicklas WJ (1996) Attenuation of excitotoxic cell swelling and GABA release by the GABA transport inhibitor SKF 89976a. Mol Chem Neuropathol 29:27–36., 1997Zeevalk GD and Nicklas WJ (1997) Activity at the GABA transporter contributes to acute cellular swelling produced by metabolic impairment in retina. Vision Res 37:3463–3470.), suggesting that overexpression of the GABA transporter can damage neurons. In the current study, Slc6a13 was up-regulated in the MPTP group compared to the control group; therefore this may be the underlying cause of neuronal intoxication. However, acupuncture stimulation at GB34 and LR3 maintained Slc6a13 gene expression at levels similar to those in control animals, suggesting that acupuncture has a neuroprotective effect.

Tryptophan hydroxylase 2 (Tph2), with a molecular weight of 56 kDa, is the predominant form of this enzyme present in brain extracts from the mesencephalic tegmentum, striatum, and hippocampus (Sakowski et al., 2006Sakowski SA, Geddes TJ, Thomas DM, Levi E, Hatfield JS and Kuhn DM (2006) Differential tissue distribution of tryptophan hydroxylase isoforms 1 and 2 as revealed with monospecific antibodies. Brain Res 1085:11–18.). Tph2 initiates serotonin synthesis in mammals with Tph1 (Alenina et al., 2009Alenina N, Kikic D, Todiras M, Mosienko V, Qadri F, Plehm R, Boye P, Vilianovitch L, Sohr R, Tenner K, et al. (2009) Growth retardation and altered autonomic control in mice lacking brain serotonin. Proc Natl Acad Sci USA 106:10332–10337.), and plays a critical role in the maintenance of brain serotonin homeostasis (Beaulieu et al., 2008Beaulieu JM, Zhang X, Rodriguiz RM, Sotnikova TD, Cools MJ, Wetsel WC, Gainetdinov RR and Caron MG (2008) Role of GSK3 beta in behavioral abnormalities induced by serotonin deficiency. Proc Natl Acad SciUSA 105:1333–1338.). Tph2-derived serotonin is involved in the regulation of behavior and autonomic pathways but is not essential for adult life (Alenina et al., 2009Alenina N, Kikic D, Todiras M, Mosienko V, Qadri F, Plehm R, Boye P, Vilianovitch L, Sohr R, Tenner K, et al. (2009) Growth retardation and altered autonomic control in mice lacking brain serotonin. Proc Natl Acad Sci USA 106:10332–10337.). However, Tph2 is known to be highly labile to oxidation (Kuhn et al., 1980Kuhn DM, Ruskin B and Lovenberg W (1980) Tryptophan hydroxylase. the role of oxygen, iron, and sulfhydryl groups as determinants of stability and catalytic activity. J Biol Chem 255:4137–4143.). Therefore, the oxidative processes that prevail in PD may cause a misfolding of Tph2 and result in the modification of serotonin function such as is seen in dopamine neurons. Tph2 oxidation inhibits its activity and leads to the formation of high-molecular-weight aggregates in a dithiothreitol-reversible manner (Kuhn et al., 2011Kuhn DM, Sykes CE, Geddes TJ, Jaunarajs KL and Bishop C (2011) Tryptophan hydroxylase 2 aggregates through disulfide cross-linking upon oxidation: Possible link to serotonin deficits and non-motor symptoms in Parkinson’s disease. J Neurochem 116:426–437.). Alteration of serotonin levels by Tph2 hyperinnervation affects the wiring of the brain and can produce long-lasting changes leading to the development of neurodevelopmental disorders such as PD (Migliarini et al., 2012Migliarini S, Pacini G, Pelosi B, Lunardi G and Pasqualetti M (2012) Lack of brain serotonin affects postnatal development and serotonergic neuronal circuitry formation. Mol Psychiatry 18:1106–1118.).

Myelin protein zero-like 2 (Mpzl2) is also known as Eva and Eva1. In human choroid plexus epithelial cells and a subset of CD4 T lymphocytes, Mpzl2 is expressed at high levels. Mpzl2 expressed in choroid plexus cells may regulate the permeability of the blood-cerebrospinal fluid (CSF) barrier (Chatterjee et al., 2008Chatterjee G, Carrithers LM and Carrithers MD (2008) Epithelial v-like antigen regulates permeability of the blood-csf barrier. Biochem Biophys Res Commun 372:412–417.), suggesting a novel mechanism of CNS immune surveillance regulation (Wojcik et al., 2011Wojcik E, Carrithers LM and Carrithers MD (2011) Characterization of epithelial v-like antigen in human choroid plexus epithelial cells: Potential role in CNS immune surveillance. Neurosci Lett 495:115–120.). In the current study, Mpzl2 was up-regulated in the MPTP group compared to controls; however, acupuncture stimulation at GB34 and LR3 maintained Mpzl2 gene expression at levels similar to those in control animals. These results also suggest that acupuncture suppresses the Mpzl2 overexpression that can be caused by MPTP neuronal loss.

The serine (or cysteine) peptidase inhibitor, clade G, member 1 (Serping1) gene encodes the serine protease inhibitor (serpin) known as C1 inhibitor. The C1 inhibitor is important for regulation of several processes, including inflammation, that are involved in maintenance of blood vessels (Shagdarsuren et al., 2008Shagdarsuren E, Bidzhekov K, Djalali-Talab Y, Liehn EA, Hristov M, Matthijsen RA, Buurman WA, Zernecke A and Weber C (2008) C1-esterase inhibitor protects against neointima formation after arterial injury in atherosclerosis-prone mice. Circulation 117:70–78.). Serping1 is up-regulated in the microglial pathway upon stimulation by interferon-gamma (IFN-gamma; (Moran et al., 2007Moran LB, Duke DC and Graeber MB (2007) The microglial gene regulatory network activated by interferon-gamma. J Neuroimmunol 183:1–6.) or lethal lipopolysaccharide-induced endotoxic shock (Liu et al., 2007Liu D, Lu F, Qin G, Fernandes SM, Li J and Davis 3rd AE (2007) C1 inhibitor-mediated protection from sepsis. J Immunol 179:3966–3972.).

Inter-alpha trypsin inhibitor, heavy chain 2 (Itih2) has also been identified during inflammation (Scavenius et al., 2011Scavenius C, Sanggaard KW, Nikolajsen CL, Bak S, Valnickova Z, Thogersen IB, Jensen ON, Hojrup P and Enghild JJ (2011) Human inter-alpha-inhibitor is a substrate for Factor XIIIa and tissue transglutaminase. Biochim Biophys Acta 1814:1624–1630.), and may contribute to the cascade of events leading to neuronal degeneration (Hirsch and Hunot 2009Hirsch EC and Hunot S (2009) Neuroinflammation in Parkinson’s disease: A target for neuroprotection? Lancet Neurol 8:382–397.; Tufekci et al., 2012Tufekci KU, Meuwissen R, Genc S and Genc K (2012) Inflammation in Parkinson’s disease. Adv Protein Chem Struct Biol 88:69–132.). Therapeutic strategies aim to down-regulate these inflammatory processes and may slow the progression of PD (Hirsch and Hunot 2009Hirsch EC and Hunot S (2009) Neuroinflammation in Parkinson’s disease: A target for neuroprotection? Lancet Neurol 8:382–397.). Therefore, the maintenance by acupuncture stimulation of expression levels of Itih2 and Serping1, which are involved in neuroinflammation, similar to those in control animals suggests that acupuncture may have a neuroprotective effect.

SLC6 (neurotransmitter transporter), member 20A (Slc6a20a) is also known as Xt3s1, Xtrp3s1, AU022428, and A730081N20Rik. Neurotransmitter transporters of the SLC6 family play an important role in the removal of neurotransmitters in brain tissue and in amino acid transport in epithelial cells (Kowalczuk et al., 2005Kowalczuk S, Broer A, Munzinger M, Tietze N, Klingel K and Broer S (2005) Molecular cloning of the mouse imino system: An Na+− and Cl—dependent proline transporter. Biochem J 386:417–422.). Either the IMINO system or the imino acid carrier supports the main load of proline transport (Munck and Munck, 1994Munck LK and Munck BG (1994) Chloride-dependent intestinal transport of imino and beta-amino acids in the guinea pig and rat. Am J Physiol 266:R997–1007.); this gene is expressed at high levels in the brain. More specifically, the IMINO system is present in the microglial cells of the brain (Kowalczuk et al., 2005Kowalczuk S, Broer A, Munzinger M, Tietze N, Klingel K and Broer S (2005) Molecular cloning of the mouse imino system: An Na+− and Cl—dependent proline transporter. Biochem J 386:417–422.), by which proline is taken up (Hubscher and Berkley, 1992Hubscher CH and Berkley KJ (1992) Glial cells: Possible determinants of neuronal uptake of tritiated proline. Neuroscience 47:737–743.; Mikulska and Lisowski, 2003Mikulska JE and Lisowski J (2003) A proline-rich polypeptide complex (prp) from ovine colostrum. Studies on the effect of prp on nitric oxide (NO) production induced by LPS in Thp-1 cells. Immunopharmacol Immunotoxicol 25:645–654.). This, in turn, influences the proline level in extracellular fluid (ECF). Proline itself has been postulated to be neurotoxic when its extracellular concentration is increased in the brain (Cohen and Nadler 1997Cohen SM and Nadler JV (1997) Proline-induced potentiation of glutamate transmission. Brain Res 761:271–282.; Nadler et al., 1988Nadler JV, Wang A and Hakim A (1988) Toxicity of l-proline toward rat hippocampal neurons. Brain Res. 456:168–172.). In the current study, Slc6a20a expression in the SN was up-regulated in the MPTP-induced mouse model of PD. Thus, a disturbance in the uptake of proline influences the extracellular concentration of proline. Increasing pro-line levels in ECF may induce neuronal cell death, suggesting that down-regulation of Slc6a20a gene expression following acupuncture may have a neuroprotective effect.

SLC6 (neurotransmitter transporter, serotonin), member 4 (Slc6a4) is also known as Htt, Sert, 5-HTT, or AI323329. Recently, it was reported that MPTP-induced dopaminergic denervation is followed by serotoninergic hyperinnervation (Hebert et al., 2005Hebert G, Mingam R, Arsaut J, Dantzer R and Demotes-Mainard J (2005) A role of IL-1 in MPTP-induced changes in striatal dopaminergic and serotoninergic transporter binding: Clues from interleukin-1 type i receptor-deficient mice. Brain Res Mol Brain Res 136:267–270.). In a 6-OHDA animal model of PD, the effects of destroying ascending dopa-mine pathways on extracellular levels of serotonin and serotonin innervation in the rat striatum were examined (Balcioglu et al., 2003Balcioglu A, Zhang K and Tarazi FI (2003) Dopamine depletion abolishes apomorphine- and amphetamine-induced increases in extracellular serotonin levels in the striatum of conscious rats: A microdialysis study. Neuroscience 119:1045–1053.). Specifically, the Slc6a4 promoter may govern the genetic risk of PD (Albani et al., 2009Albani D, Vittori A, Batelli S, Polito L, De Mauro S, Galimberti D, Scarpini E, Lovati C, Mariani C and Forloni G (2009) Serotonin transporter gene polymorphic element 5-httlpr increases the risk of sporadic Parkinson’s disease in Italy. Eur Neurol 62:120–123.). In the current study, Slc6a4 was up-regulated in the MPTP group compared to controls, and acupuncture stimulation at GB34 and LR3 maintained Slc6a4 gene expression at levels similar to those in control animals. These results indicate that acupuncture may suppress the Slc6a4 overexpression caused by MPTP intoxication.

Upper zone of growth plate and cartilage matrix associated (Ucma) is a highly conserved tyrosine-sulphated secreted protein with a molecular weight of 17 kDa that is expressed by juvenile chondrocytes (Tagariello et al., 2008Tagariello A, Luther J, Streiter M, Didt-Koziel L, Wuelling M, Surmann-Schmitt C, Stock M, Adam N, Vortkamp A and Winterpacht A (2008) Ucma - a novel secreted factor represents a highly specific marker for distal chondrocytes. Matrix Biol 27:3–11.). Ucma may be involved in the negative control of the osteogenic differentiation of osteochondrogenic precursor cells in peripheral zones of fetal cartilage and at the cartilage-bone interface (Surmann-Schmitt et al., 2008Surmann-Schmitt C, Dietz U, Kireva T, Adam N, Park J, Tagariello A, Onnerfjord P, Heinegard D, Schlotzer-Schrehardt U, Deutzmann R, et al. (2008) Ucma, a novel secreted cartilage-specific protein with implications in osteogenesis. J Biol Chem 283:7082–7093.). An imbalance in isoform expression may, therefore, be involved in skeletal pathology (Le Jeune et al., 2010Le Jeune M, Tomavo N, Tian TV, Flourens A, Marchand N, Camuzeaux B, Mallein-Gerin F and Duterque-Coquillaud M (2010) Identification of four alternatively spliced transcripts of the ucma/grp gene, encoding a new gla-containing protein. Exp Cell Res 316:203–215.). PD patients have been reported to have a significantly increased risk of fracture (Sato et al., 2001Sato Y, Kaji M, Tsuru T and Oizumi K (2001) Risk factors for hip fracture among elderly patients with Parkinson’s disease. J Neurol Sci 182:89–93.; Pouwels et al., 2013Pouwels S, Bazelier MT, de Boer A, Weber WE, Neef C, Cooper C and de Vries F (2013) Risk of fracture in patients with Parkinson’s disease. Osteoporos Int 24:2283–2290.), which may be caused by an imbalance in Ucma expression. In the current study, Ucma expression was up-regulated in the MPTP group compared to the control group, and acupuncture stimulation at GB34 and LR3 maintained Ucma expression at levels similar to those in control animals.

Retinol dehydrogenase 9 (Rdh9) is a short-chain dehydrogenase/reductase and converts 9-cis-retinol into 9-cis-retinal and 3alpha-androstanediol into dihydrotestosterone (Zhuang et al., 2002Zhuang R, Lin M and Napoli JL (2002) Cis-retinol/androgen dehydrogenase, isozyme 3 (crad3): Ashort-chain dehydrogenase active in a reconstituted path of 9-cis-retinoic acid biosynthesis in intact cells. Biochemistry 41:3477–3483.). Widespread Rdh9 function in steroid or retinoid metabolism begins mid-embryogenesis (Hu et al., 2007Hu P, Zhang M and Napoli JL (2007) Ontogeny of rdh9 (crad3) expression: Ablation causes changes in retinoid and steroid metabolizing enzymes, but RXR and androgen signaling seem normal. Biochim Biophys Acta 1770:694–705.), and may be an important link between neuroactive steroids and neurodegenerative disorders (Melcangi et al., 2012Melcangi RC, Caruso D, Levandis G, Abbiati F, Armentero MT and Blandini F (2012) Modifications of neuroactive steroid levels in an experimental model of nigrostriatal degeneration: Potential relevance to the pathophysiology of Parkinson’s disease. J Mol Neurosci 46:177–183.). Moreover, testosterone has been reported to be neurotoxic to dopaminergic neurons (Cunningham et al., 2009Cunningham RL, Giuffrida A and Roberts JL (2009) Androgens induce dopaminergic neurotoxicity via caspase-3-dependent activation of protein kinase Cdelta. Endocrinology 150:5539–5548.). The up-regulation of Rdh9 in the MPTP-treated group relative to the control group is thought to be one aspect of this consequence. However, further evidence is needed to confirm this, and the maintenance of Rdh9 expression following acupuncture may have neuro-protective effects.

Down-up regulated genes by MPTP and acupuncture

Of the 255 probes down-regulated in the SN region compared to controls, 17 (two annotated genes) were exclusively up-regulated following acupuncture at acupoints but not at non-acupoints (“Down-up”; Table 3).

The function of the RIKEN cDNA 4921530L21 gene (4921530L21Rik) has yet to be characterized. The function of the predicted gene 13931 (Gm13931) is known as olfactory receptor-related genes. The olfactory system may be particularly suitable route for the penetration of xenobiotic agents into the CNS, as was shown in an intranasal MPTP model (Prediger et al., 2009Prediger RD, Rial D, Medeiros R, Figueiredo CP, Doty RL and Takahashi RN (2009) Risk is in the air: An intranasal MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) rat model of Parkinson’s disease. Ann N Y Acad Sci 1170:629–636., 2010Prediger RD, Aguiar Jr. AS, Rojas-Mayorquin AE, Figueiredo CP, Matheus FC, Ginestet L, Chevarin C, Bel ED, Mongeau R, Hamon M, et al. (2010) Single intranasal administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in C57BL/6 mice models early preclinical phase of Parkinson’s disease. Neurotox Res 17:114–129.). Thus, olfactory receptor-related genes may be related to the pathological mechanisms of MPTP neurotoxicity.

Signal pathway analysis

Pathway analyses for the 22 probes that were up-regulated by MPTP intoxication (vs. CTL) and down-regulated by acupuncture at acupoints (vs. MPTP), as well as for the 17 probes down-regulated by MPTP intoxication (vs. CTL) and up-regulated by acupuncture at acupoints (vs. MPTP; Table 4) were performed. Cdh1, which is “down-up” regulated by MPTP and acupuncture stimulation at acupoints, was involved in “thyroid, endometrial, bladder cancer”, “bacterial invasion of epithelial cells”, and “melanoma” at significant levels according to the over-representation analysis (ORA). However, “pathways in cancer”, “adherens junction” and “cell adhesion molecules (CAMs)” were not significant. Tph2 was involved in “tryptophan metabolism” and “serotonergic synapse” at significant levels. Slc6a4 was involved in “serotonergic synapse” at a significant level. Serping1 was involved in “complement and coagulation cascades” and “pertussis” at significant levels. Slc6a13 was involved in “GABAergic synapse”, albeit not significantly so. Gm13931 was involved in “olfactory transduction” and was “down-up” regulated by MPTP and acupuncture stimulation at acupoints, even if not significant according to the ORA.

These findings demonstrate that both MPTP and acupuncture at acupoints influence Cdh1, Tph2, Slc6a4, Serping1, Slc6a13, and Gm13931 expression in the indicated pathways in the SN region.

Validation of gene expression changes in the SN region following MPTP and acupuncture at acupoints

To validate the microarray data, representative genes were selected and their expression levels in the SN region were evaluated by real time RT-PCR (Figure 3). Expression levels of Rdh9, Slc6a4, Tph2 and Ucma, which were “up-down” regulated in the microarray analysis, also showed “up-down” regulation in real time RT-PCR (Figure 3). These findings demonstrate that the regulation patterns in the SN region regulated by MPTP and acupuncture at acupoints determined by real-time RT-PCR correlate with those determined using microarrays.

Acupuncture stimulation attenuates the reduction of TH induced by 6-OHDA intoxication (Park et al., 2003Park HJ, Lim S, Joo WS, Yin CS, Lee HS, Lee HJ, Seo JC, Leem K, Son YS, Kim YJ, et al. (2003) Acupuncture prevents 6-hydroxydopamine-induced neuronal death in the nigrostriatal dopaminergic system in the rat Parkinson’s disease model. Exp Neurol 180:93–98.) and MPTP (Kang et al., 2007Kang JM, Park HJ, Choi YG, Choe IH, Park JH, Kim YS and Lim S (2007) Acupuncture inhibits microglial activation and inflammatory events in the mptp-induced mouse model. Brain Res 1131:211–219.; Choi et al., 2011bChoi YG, Yeo S, Hong YM and Lim S (2011b) Neuroprotective changes of striatal degeneration-related gene expression by acupuncture in an mptp mouse model of Parkinsonism: Microarray analysis. Cell Mol Neurobiol 31:377–391.) in nigrostriatal dopaminergic neurons. Behavioral tests demonstrated that acupuncture stimulation improves motor dysfunction in a 6-OHDA Parkinsonism model by ∼87.7% (Park et al., 2003Park HJ, Lim S, Joo WS, Yin CS, Lee HS, Lee HJ, Seo JC, Leem K, Son YS, Kim YJ, et al. (2003) Acupuncture prevents 6-hydroxydopamine-induced neuronal death in the nigrostriatal dopaminergic system in the rat Parkinson’s disease model. Exp Neurol 180:93–98.). The SN is a crucial aspect of the motor circuit (Brazhnik et al., 2012Brazhnik E, Cruz AV, Avila I, Wahba MI, Novikov N, Ilieva NM, McCoy AJ, Gerber C and Walters JR (2012) State-dependent spike and local field synchronization between motor cortex and substantia nigra in hemiparkinsonian rats. J Neurosci 32:7869–7880.; Gaugler et al., 2012Gaugler MN, Genc O, Bobela W, Mohanna S, Ardah MT, El-Agnaf OM, Cantoni M, Bensadoun JC, Schneggenburger R, Knott GW, et al. (2012) Nigrostriatal overabundance of alpha-synuclein leads to decreased vesicle density and deficits in dopamine release that correlate with reduced motor activity. Acta Neuropathol 123:653–669.) and is one of the areas damaged most markedly in PD (Castro et al., 2013Castro AA, Wiemes BP, Matheus FC, Lapa FR, Viola GG, Santos AR, Tasca CI and Prediger RD (2013) Atorvastatin improves cognitive, emotional and motor impairments induced by intranasal 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration in rats, an experimental model of Parkinson’s disease. Brain Res 1513:103–116.; Fahim et al., 2013Fahim MA, Shehab S, Nemmar A, Adem A, Dhanasekaran S and Hasan MY (2013) Daily subacute paraquat exposure decreases muscle function and substantia nigra dopamine level. Physiol Res 62:313–321.), particularly relative to dystonia (Truong et al., 2009Truong L, Brooks D, Amaral F, Henderson JM and Halliday GM (2009) Relative preservation of thalamic centromedian nucleus in parkinsonian patients with dystonia. Mov Disord 24:2128–2135.).

The neuroprotective effects of acupuncture have been confirmed at a genetic level. The microarray results indicate that Slc6a4 and Tph2, which are involved in PD, and Mpzl2 and Serping1, which are involved in inflammation, are primary causes of neurodegenerative disorders (Drouin-Ouellet and Cicchetti, 2012Drouin-Ouellet J and Cicchetti F (2012) Inflammation and neuro-degeneration: The story ‘retolled’. Trends Pharmacol Sci 33:542–551.; Pradhan and Andreasson 2013Pradhan S and Andreasson K (2013) Commentary: Progressive inflammation as a contributing factor to early development of Parkinson’s disease. Exp Neurol 241:148–155.), and their expression can be maintained at similar to normal levels by acupuncture. Therefore, the effect of MPTP intoxication on gene expression in the SN region may be ameliorated by acupuncture at acupoints. This suggests an attenuating effect of acupuncture on the degeneration of dopaminergic neuron-like cells by MPTP in the SN. Previous research employing a mouse model of acute MPTP Parkinsonism has demonstrated that acupuncture treatment suppressed genes related to cytokine-cytokine receptor interaction and oxidative phosphorylation pathways (Hong et al., 2010Hong MS, Park HK, Yang JS, Park HJ, Kim ST, Kim SN, Park JY, Song JY, Jo DJ, Park SW, et al. (2010) Gene expression profile of acupuncture treatment in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinson’s disease model. Neurol Res 32(Suppl 1):74–78.). Cellular responses to deleterious events like oxidative stress and cytokine receptor-mediated apoptosis might eventually lead to dopaminergic cell death and hence disease progression. Oxidative stress is an early event that may directly kill dopaminergic neurons (Hong et al., 2010Hong MS, Park HK, Yang JS, Park HJ, Kim ST, Kim SN, Park JY, Song JY, Jo DJ, Park SW, et al. (2010) Gene expression profile of acupuncture treatment in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinson’s disease model. Neurol Res 32(Suppl 1):74–78.). Moreover, the current study showed that at the chronic stage, the expression of genes involved in PD was modulated by acupuncture stimulation. These results suggest that a variety of genes may be related to the effect of acupuncture on the treatment of PD.

Further studies of the association between gene expression and motor function following acupuncture are warranted, as the investigated genes may mediate the protective effects of acupuncture on SN-mediated motor function. Moreover, the regulation of these genes following acupuncture and their influence on the SN via the involvement of abnormal motor circuits should be clarified.

Conclusions

We investigated gene expression changes in the SN using a whole-transcript GeneChip microarray following acupuncture stimulation at acupoints GB34 and LR3 in an MPTP-induced Parkinsonism model. Our data suggest that acupuncture at these acupoints attenuates the decrease in TH in the SN region, while acupuncture at non-acupoints did not suppress this decrease. Compared to the control group, 22 probes (10 annotated genes: Cdh1, Itih2, Mpzl2, Rdh9, Serping1, Slc6a13, Slc6a20a, Slc6a4, Tph2, and Ucma) were up-regulated in the MPTP group and were exclusively down-regulated by acupuncture at acupoints but not at non-acupoints. Additionally, compared to the control group, 17 probes (two annotated genes; 4921530L21Rik and Gm13931) were down-regulated in the MPTP group and were exclusively up-regulated after acupuncture at acupoints but not at non-acupoints. Therefore, these 39 probes (12 annotated genes) may be responsible for the protective effect of acupuncture in the SN following MPTP-induced impairment.

Supplementary Material

This material is available as part of the online version of this article from http://www.scielo.br/gmb.

This work was supported by a National Research Foundation of Korea grant funded by the Korean Government [MEST] (No. 2007-0054931, NRF-2014R1A1A1004100). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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