Materials & Methods
Discussion & Conclusion
Development of Midbrain Dopamine Neurons: Role of Epigenetic Factors
Contact Person: Umberto di Porzio (firstname.lastname@example.org)
Materials and Methods
ANIMALS AND DISSECTIONS:
Timed pregnant Sprague Dawley rats or DC1 mice (Charles River Milano) were used for cell cultures or cell grafts. Male C57BL/6N mice were used as hosts in transplantation experiments. Insemination was confirmed by vaginal plug and considered as E0.
CELL PREPARATION FOR CULTURES AND GRAFTS:
Cells were dissociated from E13 or E16 rat or mouse ventral mesencephalon, E16 striatum, E16 parietal cortex or E18 hypothalamus. Cell were cultured as previously described (11, 12). Cells were plated at a density of 50.000 per square centimeter in standard culture conditions, or 18,000 per square centimeter when basic FGF was used to induce neuroblast proliferation. Cell were grown in MEM/F12 medium supplemented with glucose to reach 33 mM, with or without addition of 7% serum (HyClone Laboratories, Inc., Milano) or N2 or B27 supplements (GIBCO BRL, Milano) (13). In some experiments transwell plates were used (Costar) to separate cells by a membrane with 0.4 millimicron pore size. 10 micromolar cytosine b-D-arabinofuranoside (Sigma) was added to cultures grown in the presence of serum to inhibit non-neuronal cell proliferation.
RNA was isolated from tissue samples or cultures by using guanidium isothiocyanate or tri-reagent kit (Sigma). The yield and the integrity of RNA were determined by spectrophotometrical measurement and agarose gel electrophoresis. The highly sensitive RT-PCR assay allows to detect relatively small changes in the levels of the gene transcripts, thus enabling to study genes with low level of expression. Optimized conditions were as previously described. Various sets of primer pairs were used in the same reaction tube to co-amplify cDNA. Co-amplification of two, three or four fragments resulted in similar yield for each PCR product. This procedure allows to compare the expression of various genes and overcome the inherent variations present within any individual PCR by normalizing to hypoxantine-phosphoribosyl-transferase (HPRT) mRNA level, used as an internal standard (14). Amplified products were separated by 1.5 % agarose gel electrophoresis and exposed to a PhosphorImager screen and scanned (Molecular Dynamics, Sunnyvale, CA). Data are expressed as the ratio between each amplified product and the co-amplified HPRT. Identity of amplified fragments was confirmed by digestion with restriction enzymes or sequence analysis. To avoid amplification of possible contaminating genomic DNA, most primers were designed in order to span at least one intron. When this was not possible, RNA samples were DNased before reverse transcription.
Animals were anesthetized with sodium pentobarbital (50 mg/Kg) and sacrificed by transcardiac perfusion with 2% paraformaldehyde in 0.1 M PBS pH 7.4, containing 1.5% lysine and 0.2% sodium periodate, the brains removed, postfixed and sectioned at the cryostat. To fix early embryonic tissues, embryos were removed from the wombs and immediatly immersed in 5% acrolein in PBS (15). Cell cultures were fixed in 4% paraformaldehyde for 30-60 min. at room temperature, rinsed in PBS and permeabilized with 0.2% triton X-100. Tyrosine hydroxylase-like immunoreactivity was detected with a rabbit anti-TH antibody (Eugene Tech Allandale, NJ) (dil. 1:1000) and reveled by avidin-biotin method (16) or fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (17, 12).
ANIMAL TREATMENTS AND BEHAVIORAL TESTS:
8-9 weeks old C57BL/6N mice were injected in peritoneum with acetaldehyde (250 mg/Kg) prior to MPTP hydrochloride treatment (36 mg/Kg, corresponding to 30 mg/Kg free base, RBI, Weyland, MA). One month after treatment, the level of nigrostriatal pathway lesion was tested by measuring with an electronic meter the motor activity of treated animals after administration of 0.25 mg/kg apomorphine. The individuals that showed locomotor activity 8-10 fold greater that controls were selected for receiving grafts or be sham-operated. At various times after unilateral grafting or sham operation, animals were injected with 2 mg/kg amphetamine and turning behavior was examined to evaluate DA functional recovery. Animals were sacrificed four months after operation and analyzed by immunocytochemistry.
2 microliters of saline alone (sham-controls) or containing 3 million cells were injected by peristaltic pump into the host right striatum under stereotactic coordinates (2 mm lateral, 0.2 mm rostral to Bregma, 3.5 mm below the skull) in two min. After injection the needle was left in place for four min. and then withdrawn in four min.
Analysis of variance was used to compare data (Scheffe F-test).
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