Elsevier

Neuroscience

Volume 108, Issue 2, 10 December 2001, Pages 273-284
Neuroscience

Additive effect of glial cell line-derived neurotrophic factor and neurotrophin-4/5 on rat fetal nigral explant cultures

https://doi.org/10.1016/S0306-4522(01)00418-3Get rights and content

Abstract

Transplantation of embryonic dopaminergic neurons is an experimental therapy for Parkinson’s disease, but limited tissue availability and suboptimal survival of grafted dopaminergic neurons impede more widespread clinical application. Glial cell line-derived neurotrophic factor (GDNF) and neurotrophin-4/5 (NT-4/5) exert neurotrophic effects on dopaminergic neurons via different receptor systems. In this study, we investigated possible additive or synergistic effects of combined GDNF and NT-4/5 treatment on rat embryonic (embryonic day 14) nigral explant cultures grown for 8 days.

Contrary to cultures treated with GDNF alone, cultures exposed to NT-4/5 and GDNF+NT-4/5 were significantly larger than controls (1.6- and 2.0-fold, respectively) and contained significantly more protein (1.6-fold). Treatment with GDNF, NT-4/5 and GDNF+NT-4/5 significantly increased dopamine levels in the culture medium by 1.5-, 2.5- and 4.7-fold, respectively, compared to control levels, and the numbers of surviving tyrosine hydroxylase-immunoreactive neurons increased by 1.7-, 2.1-, and 3.4-fold, respectively. Tyrosine hydroxylase enzyme activity was moderately increased in all treatment groups compared to controls. Counts of nigral neurons containing the calcium-binding protein, calbindin-D28k, revealed a marked increase in these cells by combined GDNF and NT-4/5 treatment. Western blots for neuron-specific enolase suggested an enhanced neuronal content in cultures after combination treatment, whereas the expression of glial markers was unaffected. The release of lactate dehydrogenase into the culture medium was significantly reduced for GDNF+NT-4/5-treated cultures only.

These results indicate that combined treatment with GDNF and NT4/5 may be beneficial for embryonic nigral donor tissue either prior to, or in conjunction with, intrastriatal transplantation in Parkinson’s disease.

Section snippets

Preparation of cultures

Under deep pentobarbital anesthesia, embryos (embryonic day (E) 14) were removed by Cesarean section from pregnant Sprague–Dawley rats (BRL Biological Research, Füllinsdorf, Switzerland) and killed by decapitation (all efforts were made to minimize both the suffering and number of animals used). From the isolated brains the ventral mesencephali were dissected out according to standard techniques (Dunnett and Björklund, 1992), and grown as FFRT cultures (Spenger et al., 1994). In brief, each

Effect of growth factor treatment on culture volume and protein content

Cultures from all experimental groups showed a steady increase in volume during the culture period as observed by routine inspection. At day 4, the cultured mesencephalic tissue had formed small spheres, and at day 8 the culture diameter ranged between 0.9 and 1.5 mm. At that time-point, cultures treated with NT-4/5 or GDNF+NT-4/5 were significantly larger than controls [59% (P<0.05) and 101% (P<0.01), respectively], whereas GDNF-treated cultures were not different from controls (P>0.05; Table 1

Discussion

Intracerebral transplantation into severely diseased parkinsonian patients has provided promising results in several clinical trials, but suboptimal survival or differentiation of grafted dopaminergic neurons as well as ethical concerns and the limited availability of embryonic human donor tissue prevent a more widespread application as described in several papers (Lindvall et al., 1990, Freed et al., 1992, Freeman et al., 1995, Olanow et al., 1996, Kordower et al., 1998, Tabbal et al., 1998,

Acknowledgements

The expert technical assistance of Sandra Krebs, Tanja Bosnjak, and Inge Holst is gratefully acknowledged. We also wish to express our gratitude to Dr. Christian Spenger, Stockholm, Sweden for help with the HPLC analysis, Dr. Lars Korsholm, Odense, Denmark for statistical advice, and Dr. Anthony Castro, Marywood, USA for critical comments on the manuscript. This research was supported by the Swiss National Science Foundation (Grant No. 31-52947.97), Desirée and Niels Ydes Foundation, the Danish

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