ABSTRACT: We have previously detected cyclin D1-initiated and p53-related (PERP; p53 apoptosis-associated target) cell death in a striatal 6-hydroxydopamine (6-OHDA)-treated Parkinsonian mouse model by adaptor-tagged competitive PCR (ATAC-PCR) (Synapse 51, 279-286, 2004). In order to establish the mechanism of dopaminergic cell death in Parkinsonâs disease, various Parkinsonian animal models should be studied. Therefore, the time-course alteration of gene expression in the substantia nigra pars compacta was investigated after a single administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in common marmosets. In the MPTP model, gene expression of neither cyclin D1 nor PERP was altered. There were also few common alterations in gene expression between the MPTP and 6-OHDA models. This suggests that the mechanism of cell death may differ between these models, and extensive study in various Parkinsonian models should be performed to elucidate the mechanisms of dopaminergic cell death in Parkinsonâs disease. Experiment Overall Design: Materials and methods Experiment Overall Design: 1. Animals Experiment Overall Design: Five 3-year-old male common marmosets (Callithrix jacchus) weighing 350â400 g were used. A single marmoset was used as a vehicle-treated control, with the rest being administered MPTP. A microarray experiment and an immunohistochemical study were performed in the same animals. They were colonized in the Center for Life Science Resources in Kagoshima University, Japan. Animals were housed with free access to standard food in an air-conditioned room under a constant 12 h light/dark cycle (lights on at 7:00 a.m.) at a temperature of 25â27°C and 45â55% humidity. All efforts were made to minimize animal suffering, and to reduce the number of animals used. The present experiments were carried out after obtaining permission from the Committee of Animal Experimentation, Kagoshima University Graduate School of Medical and Dental Sciences, Japan. Experiment Overall Design: 2. MPTP administration Experiment Overall Design: A single dose of MPTP was administered intravenously (i.v.) to the animals in order to examine the time-course alteration in gene expression. As in our previous study, the dose of MPTP was 2.5 mg/kg (Nomoto et al., 1997). At this dose, animals exhibit immobility during an entire day, while doses greater than 2.5 mg/kg can induce death. MPTP hydrochloride (Sigma, St. Louis, MO, USA) was dissolved in 0.9% saline, and administered under intraperitoneal pentobarbital anesthesia (40 mg/kg). Under deep anesthesia with pentobarbital, the animals were decapitated either at 2 h, 6 h, 1 day or 14 days after MPTP administration. All experiments using MPTP were performed in accordance with safety guidelines (Przedborski et al., 2001). Experiment Overall Design: 3. Laser capture microdissection Experiment Overall Design: The midbrain of each marmoset was dissected on an ice-cold glass plate, and mounted on a specimen block with O.C.T. compound (Tissue-Tek, Sakura Finetechnical, Tokyo, Japan). The midbrains were frozen by covering them with very finely powdered dry ice. Sections (8 μm thickness) were cut on a cryostat (Microm HM500S, Carl Zeiss Japan, Tokyo, Japan), and mounted on ice-cold, membrane-coated glass slides (Carl Zeiss Japan). The membranes on the glass slides were coated with poly-L-lysine (P8920; Sigma). Immediately after mounting, slides were cooled on dry ice and stored at â80°C for less than 3 days. Slides were fixed in ethanol/acetate solution (19:1) at 4°C for 3 min, and washed with PBS at 4°C for 1 min. Slides were stained with 0.1% toluidine blue at room temperature for 10 s, washed with PBS at 4°C for 10 s, and then air-dried for 5 min. Immediately after staining, slides were processed for laser capture microdissection (LCM). Experiment Overall Design: A Leica AS LMD (Leica Microsystems, Wetzlar, Germany) was used for LCM. SNc sections corresponded to level A4.5 of the brain atlas (Stephan et al., 1980). These sections contained transverse sections of the oculomotor nerve. A strip of the unilateral SNc was dissected (Fig. 1). The SNcs from two slides were collected and placed in a PCR tube containing 10 μl extraction buffer of the PicoPure RNA Isolation Kit (Arcturus Bioscience, Mountain View, CA, USA). Experiment Overall Design: 4. Microarray experiments Experiment Overall Design: Total RNA was extracted by the PicoPure RNA Isolation Kit, and eluted in approximately 10 μl buffer, according to the manufacturerâs protocol. In order to obtain a sufficient amount of amplified RNA (aRNA) for the microarray experiments, total RNA was amplified using two rounds of in vitro transcription. The first round of amplification was performed using the RiboAmp HS RNA Amplification Kit (Arcturus). The second round of amplification used the Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies, Palo Alto, CA, USA) and cyanine-3 or cyanine-5 labeling. The labeled aRNA was hybridized with Agilent whole human genome oligo microarray G4112A. In order to correct the buffer composition in the second round of amplification using the Low RNA Input Fluorescent Linear Amplification Kit, cDNA (11 μl) that was purified in the round 2 procedure by the RiboAmp HS RNA Amplification Kit, was added to the modified transcription buffer of the Agilent kit. Each sample contained 4 μl of 5 ï´ first strand buffer, 20 μl of 4 ï´ transcription buffer, 6 μl 0.1 M DTT, 8 μl NTP mix, 6.4 μl 50% PEG, 0.5 μl RNase inhibitor (RNase Out; Invitrogen, Carlsbad, CA, USA), 0.6 µl inorganic pyrophosphatase, 0.8 μl T7 RNA polymerase, 2.4 μl cyanine (PerkinElmer Life Sciences, Boston, MA, USA; cyanine-3 for controls and cyanine-5 for MPTP-treated marmosets), and 20.3 μl DW. Labeled aRNA was synthesized according to the instructions for the Agilent kit. Experiment Overall Design: An Agilent 2100 bioanalyzer and an RNA 6000 Pico LabChip Kit were used to examine the length distribution of labeled aRNAs. A preliminary experiment using the protocol found in the appendices of the HistoGene LCM Immunofluorescence Staining Kit (Arcturus) was performed to determine the degree of degradation of total RNA that occurred during tissue staining.
