Animals and treatment
Timed-pregnant Fisher F344 rats were obtained from Charles River Laboratories (Raleigh, NC). Eight-week-old male C57BL/6J mice, weighing 25–28 g, were maintained on a 12:12 h light:dark cycle and fed ad libitum. PHOX-deficient (gp91 PHOX-/-) and wild-type C57BL/6J (gp91 PHOX+/+) mice were obtained from the Jackson Laboratory (Bar Harbor, ME). Animals were adapted for 2 weeks to the conditions described above before experimentation. To examine the effect of FLZ on MPTP-induced neurotoxicity, mice received daily MPTP injections [15 mg/kg of MPTP.HCl, s.c.] for 6 consecutive days. From the third day on, FLZ (75 mg/kg, p.o.) was administered 30 min before every MPTP injection for the last 4 days. Housing, breeding, and experimental use of the animals were performed in strict accordance with the National Institutes of Health guidelines.
FLZ was kindly provided by Professor Xiaotian Liang in the Department of Pharmaceutical Chemistry, Institute of Materia Medica, Chinese Academy of Medical Science. It is a white powder with 99% purity. The polyclonal anti-tyrosine hydroxylase (TH) antibody was a generous gift from Dr. John Reinhard (GlaxoSmithKline, Research Triangle Park, NC). LPS (strain O111:B4) and 2', 7'-dichlorofluorescin diacetate were purchased from Calbiochem (San Diego, CA). [3H]-DA (28 Ci/mmol) was purchased from PerkinElmer Life Sciences Inc. (Boston, MA). The mouse monoclonal antibody raised against OX42 antigen was purchased from Chemicon International (Temecula, CA). Rabbit anti-p47phoxwas obtained from Millipore Corporation (Bedford MA). FITC-conjugated goat anti-rabbit IgG antibody was obtained from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA). Rabbit anti-GAPDH antibody was obtained from Abcam (Cambridge, MA). Mouse anti-gp91phoxantibody was purchased from BD Transduction Laboratories (San Jose, CA). All other reagents came from Sigma (St Louis, MO).
Primary rat mesencephalic neuronal-glial cultures were prepared as described previously . Briefly, after the ventral mesencephalic tissues were removed and dissociated by a mechanical triturating, cells were seeded at 5 × 105/well to 24-well culture plates or 1.5 × 105/well to 96-well culture plates in maintenance medium. Seven-day-old cultures were used for treatment. Immunocytochemical analysis indicated that at the time of treatment the cultures were made up of ~10% microglia, ~50% astroglia, and ~40% neurons, of which 1 ~2% were TH-immunoreactive neurons.
Primary microglia-enriched cultures were prepared from the whole brains of 1 or 2-day-old mice, as described previously . Briefly, brain tissues, devoid of meninges and blood vessels, were dissociated by mechanical trituration. The isolated cells (5 × 107) were seeded in 150 cm2 culture flasks in Dulbecco's modified Eagle medium. Upon reaching confluence (12–14 days), microglia were separated from astroglia by shaking the flasks at 180 rpm for 1 h. Purity of the microglia-enriched cultures was >98%, as determined by immunocytochemical staining.
The rat microglia HAPI cells were generous gifts from Dr. James R. Connor (Pennsylvania State University, Hershey, PA). They were maintained at 37°C in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 50 U/ml penicillin, and 50 μg/ml streptomycin in a humidified incubator with 5% CO2/95% air.
DA uptake assay
Degeneration of DA neurons was assessed by measuring the ability of cultures to take up DA. DA uptake assays were performed as previously described . Briefly, after two washes with warm Krebs-Ringer buffer the cultures were incubated for 20 min at 37°C with 1 μM [3H]-DA for DA uptake. Then the cultures were washed 3 times with ice-cold KRB, and the cells were then dissolved in 1 N NaOH. Radioactivity was determined by liquid scintillation counting. Nonspecific DA uptake, determined in the presence of mazindol (10 μM), was subtracted.
DA neurons were recognized with an anti-TH antibody, and microglia were detected with the OX-42 antibody, as described previously. For morphological analysis, images were acquired using an inverted microscope (Nikon, Tokyo, Japan) connected to a camera (DAGE-MTI, Michigan City, IN) operated with the MetaMorph software (Universal Imaging Corporation, Downingtown, PA). To quantify cell numbers, total TH positive cells in entire wells were counted by three individuals. The average of these scores was reported.
Assay for inflammatory factors
For the TNF-α assay, culture supernatant was collected after 3 h of stimulation with LPS. The concentration of TNF-α was measured with a mouse TNF-α enzyme-linked immunosorbent assay kit from Genzyme (Cambridge, MA).
The production of NO was assessed as the accumulation of nitrite in the culture supernatant, using a colorimetric reaction with the Griess reagent. The culture supernatants were collected after 24 h of stimulation with LPS and mixed with equal volumes of the Griess reagent. The absorbance at wavelength 540 nm was measured with a UV MAX kinetic microplate reader (Molecular Devices).
The production of PGE2 in the enriched microglial cultures was evaluated 24 h after LPS treatment using a PGE2 EIA kit from Cayman (Ann Arbor, MI), according to the manufacturer's instructions.
Assay for reactive oxygen species (ROS)
Production of superoxide was determined by measuring the superoxide dismutase (SOD)-inhibitable reduction of the water-soluble tetrazolium salt (WST-1) 30 min after LPS treatment . Microglia-enriched cultures in 96-well culture plates were washed twice with Hanks' Balanced Salt Solution (HBSS) without phenol red. Cultures were then incubated at 37°C for 1 h with vehicle control or FLZ in HBSS (50 μl/well). Then, 50 μl of HBSS with and without SOD (50 U/ml) was added to each well along with 50 μl of WST-1 (1 mM) in HBSS and 50 μl of vehicle or LPS (2 ng/ml). Thirty minutes later, the absorbance at 450 nm was read with a SpectraMax Plus microplate spectrophotometer (Molecular Devices, Sunnyvale, CA).
The production of intracellular ROS was measured by 2', 7'-dichlorofluorescin diacetate oxidation 2 h after LPS treatment. Microglia-enriched cultures were seeded (5 × 104) in 96-well plates and then exposed to 20 μM 2', 7'-dichlorofluorescin diacetate for 1 h, followed by pretreatment with FLZ for 1 h and treatment with HBSS containing LPS. After incubation, the fluorescence was read at the 485 nm excitation and 530 nm emission on a fluorescence plate reader. Cell-free experiments with and without FLZ were conducted to determine that the reagents themselves did not alter fluorescence.
Real-time RT-PCR analysis
The primers synthesized from Sigma Genosys were used as follows: TNF-α, forward (TCGTAGCAAACCACCAAGCA) and reverse (CCCTTGAAGAGAACCTGGGAGTA); iNOS, forward (GTGCTAATGCGGAAGGTCATG) and reverse (CGCTTCCGACTTTCCTGTCT); COX2, forward (CCAGCAGGCTCATACTGATAGGA) and reverse (GCAGGTCTGGGTCGAACTTG); GAPDH, forward (CCTGGAGAAACCTGCCAAGTAT) and reverse (AGCCCAGGATGCCCTTTAGT); and gp91, forward (CCTGCAGCCTGCCTGAATT) and reverse (AAGGAGAGGAGATTCCGACACA).
HAPI cells were treated with 10 ng/ml LPS for 24 h in the presence or absence of 10 μM FLZ. The cells were removed from culture and washed twice with cold FACS buffer (saline with 5% normal goat serum). Cells were incubated with FcR block for 30 min at 4°C and then incubated for 45 min at 4°C with PE-conjugated OX6 antibody or the appropriate isotype control antibody (BD Pharmingen). After antibody binding, cells were washed and fixed. Fluorescence was analyzed on a FACSCalibur (BD Biosciences). The mean fluorescent intensity (MFI) of experimental group was determined by subtracting the MFI of the isotype control from the MFI of the unstimulated or LPS-stimulated microglia.
Subcellular fractionation and western blot analysis
Subcellular fractionation was performed as described previously . HAPI cells, seeded in dishes at 5 × 104 cells/well, were treated with 10 ng/ml LPS for 10 min in the absence or presence of 10 μM FLZ for 1 h. The cells were then lysed in hypotonic lysis buffer, incubated on ice for 30 min, and subjected to Dounce homogenization (~20–25 strokes, tight pestle A). The lysates were loaded onto sucrose in lysis buffer and centrifuged at 1,600 g for 15 min; the supernatant above the sucrose gradient was used as the cytosolic fraction after centrifugation at 150,000 g for 30 min. The pellets, solubilized in 1% Nonidet P-40 hypotonic lysis buffer, were used as the membranous fraction. For western blot analysis, the primary antibodies included anti-p47phox(1: 2000), anti-GAPDH (1:2000) and anti-gp91phox(1:2000).
Quantitative measurements of motor coordination in mice were performed using an accelerating rota-rod (model 7650, UGO Basile, Comerio, VA, Italy). The rota-rod consisted of a plastic rod (diameter = 3 cm; length = 30 cm) partitioned off with round plates to prevent the mice from escaping from the sides of the rod. The rod was covered with smooth plastic tubing and suspended 16 cm above five plastic levers attached to timers that stop when mice land on the lever surface. The mice were placed on the rod that rotated at 3 rpm at 0 min and accelerated from 4 to 20 rpm over 5 min. Mice were oriented perpendicular to the long axis of the rod, such that the mice had to make forward walking movements to avoid falling. The latency time to fall was measured using mice 28 days after their first dose of MPTP, with each mouse experiencing three trials per day with an intertrial interval of 10 min. The average latency of each mouse was calculated.
Immunohistochemistry and cell counting in the SNpc of C57BL/6 mice
Mouse brains were cut on a horizontal sliding microtome into transverse free-floating sections, 35 μm in thickness. Six to eight brain sections of SNpc were collected at intervals of 140 μm. For immunohistochemistry, the following concentrations of antibodies were used: anti-TH (1:5000), anti-Iba-1 (1: 1000), biotinylated anti-mouse IgG (1:1000). Digital images of TH neurons in SNpc were acquired on an Olympus microscope (Olympus®, Tokyo, Japan) using an attached Polaroid digital microscope camera (Polaroid®, Cambridge, MA, USA). Twenty four consecutive brain slices (35-μm thickness), which encompassed the entire substantia nigra compacta, were collected. A normal distribution of the number of TH-IR neurons in the SNpc was constructed based on the counts of 24 slices from saline-treated mice, MPTP-treated mice and FLZ-treated mice. The distribution curves from these groups superimpose and show no difference in number and shape of the curves. Six to eight evenly spaced brain slices from different groups of animals were immunoreacted using an antibody against TH, and immunoreactive cells were counted. The distribution of cell numbers from each animal was matched with the normal distribution curve to correct for errors resulting from the cutting. Three individuals performed counting in a double-blind manner. Conclusions were drawn only when the interobserver differences were within 5%. A mean value for the number of SNpc TH neurons was then deduced by averaging the counts of 6–8 sections for each animal.
The data are presented as mean ± S.E.M. For multiple comparisons of groups, two-way ANOVA was used. Statistical significance of differences between groups was assessed using paired Student's t test, followed by Bonferroni correction using the JMP program (SAS Institute, Cary, NC, USA). A value of P < 0.05 was considered statistically significant.