A total of 24 adult male Sprague-Dawley rats (weight 250.300 g) were used. These animals received either a daily dose of 1,25-(OH)₂D₃ (n=12, 1 μg/ml at 1 ml/kg/day; Bonky, Calcitriol, Yuyu, Anyang, Korea) or a vehicle (n=12, ethanol 0.1%, 1 ml/kg; Merck, Darmstadt, Germany), injected intraperitoneally for 7 consecutive days. On the 7th day of injection, the animals were anesthetized and injected unilaterally with 6-OHDA (8μg/3μl in normal saline containing 0.2 mg/ml ascorbic acid; Merick, MA, USA) over 5 min using a Hamilton syringe into the medial forebrain bundle (coordinates: 4.3 mm posterior from bregma, 1.4 mm from the midline relative to the bregma, and 8.4 mm below the dural surface).

Locomotor activity was tested with the aid of apomorphine (0.1 mg/kg injected subcutaneously) 4 weeks after inflicting unilateral 6-OHDA lesions. Rats were killed 4 weeks after surgery, and their brains were fixed and processed for immunohistochemical staining as described previously.6 The primary antibodies used in this study were as follows: rabbit polyclonal anti-tyrosine-hydroxylase (TH) antibody (diluted 1:1000; Santa Cruz Biotechnology, CA, USA) and mouse monoclonal anti-rat-CD11b antibody (diluted 1:500; OX-42, Serotec, Oxford, UK). Immunostaining was visualized using the Envision™ detection kit (K5007, Dako, Carpinteria, CA, USA) and 3,3-diaminobenzidine (Sigma, St Louis, MO, USA) containing 0.01% H₂O₂. Coronal 30-μm-thick sections of the substantia nigra were made (coordinates: bregma-5.30 mm, interaural 3.70 mm), and measurements were made on pairs of sections separated from each other by 300 μm.7 The numbers of TH-immunoreactive neurons and activated microglia (CD11b-immunoreactive cells) were counted in each section from normal and lesioned substantia nigra pars compacta (SNpc), while viewing at a magnification of x100. Average numbers of immunoreactive cells per section were calculated. The ANOVA with two variables test was used for statistical analysis.

A total of 56 C57BL/6 mice weighing 25-30 g received a daily intraperitoneal injection of either 1,25-(OH)₂D₃ (n=28, 1μg/ml at 1 ml/kg/day; Bonky, Calcitriol) or vehicle (n=28, ethanol 0.1%, 1 ml/kg Merck) for 7 consecutive days. On the 7th day of injection, the animals also received four intraperitoneal injections of 20 mg/kg MPTP (calculated as free base, dissolved in 0.9% saline solution) at 2-h intervals. Control mice received saline, which was administered using the same administration schedule (n=16).

The mice were sacrificed, transcardial perfusion fixation was performed at 24 or 48 h after MPTP injection, and the brains were fixed and processed for immunohistochemical staining as described above. Measurements were made only for those cells that fell clearly within the confines of the substantia nigra (coordinates: bregma -3.16 mm, interaural 0.64 mm) in three coronal sections that were 30-μm thick and separated from each other by 300 μm.8

To investigate the temporal pattern of expression of the key cytokines involved in inflammation and immune responses (IFNγ and TNFα), gene expression was quantified using a modification of a previously described reverse transcriptase (RT)-polymerase chain reaction (PCR) method.9 Animals were killed by spinal cord dislocation and then decapitated at 4, 12, 24, or 48 h after MPTP intoxication (3 animals for each time point). RNA was prepared from the substantia nigra, as described previously. Briefly, after the RT reaction the cDNA was amplified using the following primers: IFNγ, forward: AGC GGC TGA CTG AAC TCA GAT TGT AG (26-mer), reverse: GGTC ACA GTT TTC AGC TGT ATA GGG (25-mer); TNFα, forward: GGC AGG TCT ACT TTG GAG TCA TTG C (25-mer), reverse: ACA TTC GAG GCT CCA GTG AAT TCG G (25-mer); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), forward: TGA AGG TCG GAG TCA ACG GAT TTG GT (26-mer), reverse: CAT GTG GGC CAT GAG GTC CAC CAC (24-mer). As a control for genomic contamination, a sample without RT was added to each PCR analysis. To eliminate sample-to-sample differences in RNA extraction and conversion to cDNA, we amplified the housekeeping gene GAPDH. The thermal cycling parameters were as follows: 35 cycles of 95℃ for 2 min, 94℃ for 50 s, 60℃ for 50 s, and 72℃ for 1 min; 72℃ for 5 min for TNFα and INFγ and 30 cycles of 94℃ for 10 min, 94℃ for 30 s, 57℃ for 30 s, and 72℃ for 30 s; 72℃ for 10 min for GAPDH. PCR products were separated on 1% agarose gels stained with ethidium bromide and visualized with the aid of UV light and a camera linked to an image analyzer. The results are expressed in relative units, as determined by normalization of the optical density of each band to that of the GAPDH band. Three cytokine PCR assays were performed for each group and all results were analyzed statistically using the Mann-Whitney U test.

The loss of TH-immunoreactive cells in the ipsilateral SNpc reached 58% at 4 weeks after 6-OHDA injection relative to the noninjected side (lesioned substantia nigra, 48.27±12.42 cells, mean ±SD; nonlesioned substantia nigra, 114.12±9.84, P<0.01, n=12). 1,25-(OH)₂D₃ pretreatment protected against 6-OHDA-mediated damage in the SNpc (lesioned SNpc, 77.93±9.52; nonlesioned, 118.34±12.90, P<0.05, n=12; Fig. 1A). In 1,25-(OH)₂D₃-pretreated, 6-OHDA-injected rats, only a few immunoreactive activated microglia were observed in the ipsilateral SNpc (12.24±11.31). In contrast, vehicle-treated, 6-OHDA-injected rats exhibited a more intense expression of activated microglia along the lesioned SNpc (65.38±9.82, P<0.01; Fig. 1B).

MPTP exposure led to a more than 54% loss of TH-positive neurons (117.3±24.6 vs. 252.6±16.8, P<0.01, 24 h, n=8; and 104.6±25.2 vs. 258.4±22.3, P<0.01, 48 h, n=8 Fig. 1C). Pretreatment with 1,25-(OH)₂D₃ protected this reduction in nigral TH immunoreactivity. The mean number of TH-positive neurons was significantly higher in the 1,25-(OH)₂D₃-treated groups than in the MPTP- and vehicle-treated groups (214.7±12.6 vs. 117.3±24.6, P<0.01, 24 h, n=8; and 188.6±31.3 vs. 104.6±25.2; P<0.01, 48 h, n=8 Fig. 1C). In 1,25-(OH)₂D₃-treated mice, there was an approximately 73% inhibition of microglia activation in the number of microglial profiles at 24 h after MPTP injection compared with those MPTP mice that were not given 1,25-(OH)₂D₃ (1,25-(OH)₂D₃ group, 21.22±13.33; ethanol group, 77.32±12.21, P<0.01; Fig. 1D).

RT-PCR analysis revealed a similar pattern in the kinetics of INFγ and TNFα mRNA expression following MPTP injection (Fig. 2). A rapid increase in the amount of mRNA for INFγ and TNFα was observed between 4 and 24 h after MPTP injection. Comparison with the vehicle-treated group revealed that pretreatment with 1,25-(OH)₂D₃ protected against the overexpression of proinflammatory cytokines.