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  • Title: Improved survival of embryonic porcine dopaminergic neurons in coculture with a conditionally immortalized GDNF-producing hippocampal cell line.
    Author: Meyer M, Johansen J, Gramsbergen JB, Johansen TE, Zimmer J.
    Journal: Exp Neurol; 2000 Jul; 164(1):82-93. PubMed ID: 10877918.
    Abstract:
    Transplantation of embryonic nigral tissue is used as an experimental therapy for patients with Parkinson's disease but is hampered by a limited survival rate of dopaminergic neurons. Glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor for nigrostriatal dopaminergic neurons, and the present in vitro study aimed at improving the survival of dopaminergic neurons in porcine mesencephalic brain slice cultures by adding transfected, immortalized, temperature-sensitive GDNF-releasing HiB5 cells (HiB5-GDNF). Embryonic (E27/28) porcine ventral mesencephalic brain slices were placed on membrane inserts in six-well plates with serum-containing medium, and HiB5-GDNF, nontransfected HiB5 cells (HiB5-control), or green fluorescent protein-producing HiB5 cells (HiB5-GFP) were seeded onto each tissue slice. The concentration of GDNF in the coculture medium was 0.49 +/- 0.13 ng/ml at day 9 and 0. 22 +/- 0.05 ng/ml at day 19 (mean +/- SEM) as measured by GDNF ELISA. The decrease in release of GDNF over time was paralleled by a gradual reduction in the number of HiB5-GFP cells expressing the reporter gene (EGFP). At day 12, HPLC analysis revealed that medium from HiB5-GDNF cocultures contained 2.0 times more dopamine than medium from HiB5-control cocultures. At day 21 there was 1.6 times more dopamine. Similar results were obtained for the dopamine metabolite 3,4-dihydroxyphenylacetic acid. At day 21, cell counts showed that HiB5-GDNF cocultures contained 1.5 times more tyrosine hydroxylase immunoreactive neurons than HiB5-control cocultures, which must be compared with a 1.8 fold increase after chronic treatment with rhGDNF (10 ng/ml). In conclusion, the better survival of HiB5-GDNF cocultures is promising for the generation of effective cell lines for local delivery of neurotrophic factors to intracerebral nigral grafts.
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