Jakobsen B, Gramsbergen J B, Møller Dall A, Rosenblad C, Zimmer J
Anatomy & Neurobiology, Institute of Medical Biology, University of Southern Denmark, Winsløwparken 21, 5000 Odense C, Denmark.
Eur J Neurosci. 2005 Jun;21(11):2939-48. doi: 10.1111/j.1460-9568.2005.04138.x.
We characterized organotypic ventral mesencephalic (VM) cultures derived from embryonic day 12 (E12) mice (CBL57/bL6) in terms of number of dopaminergic neurons, cell soma size and dopamine production in relation to time in vitro and tested the effects of 1-methyl-4-phenylpyridinium (MPP(+)) and glial derived neurotrophic factor (GDNF) to validate this novel culture model. Dopamine production and dopaminergic neuron soma size increased dramatically with time in vitro, whereas the number of dopamine neurons declined by approximately 30% between week 1 and week 2, which was further reduced after week 4. GDNF treatment (100 ng/mL) increased dopaminergic neuron soma size (up to 43%) and DOPAC production (approximately three-fold), but not the number of dopamine neurons in control cultures. One-week-old cultures were more vulnerable to MPP(+), than three-week-old cultures. The EC(50) for dopamine depletion after 2 days exposure and 15 days of recovery were 0.6 and 7 microm, respectively. Both pre-treatment and post-treatment with GDNF are important to obtain maximal protection against MPP(+) toxicity. In one-week-old cultures (5 microm MPP(+), 2 days) GDNF provided potent neuroprotection with dopamine contents reaching control levels and number of tyrosine hydroxylase (TH)(+) cells up to 80% of control, but in three-week-old cultures (10 microm MPP(+), 2 days) the protective potential of GDNF was markedly reduced. Long recovery periods after MPP(+) exposure are required to distinguish between reversible or irreversible toxic and/or trophic effects.
我们对源自胚胎第12天（E12）小鼠（CBL57/bL6）的器官型腹侧中脑（VM）培养物进行了表征，分析了多巴胺能神经元数量、细胞体大小以及多巴胺生成与体外培养时间的关系，并测试了1-甲基-4-苯基吡啶鎓（MPP(+)）和胶质细胞源性神经营养因子（GDNF）的作用，以验证这种新型培养模型。多巴胺生成和多巴胺能神经元细胞体大小随体外培养时间显著增加，而多巴胺能神经元数量在第1周和第2周之间下降了约30%，第4周后进一步减少。GDNF处理（100 ng/mL）增加了多巴胺能神经元细胞体大小（高达43%）和3,4-二羟基苯乙酸（DOPAC）生成（约三倍），但未增加对照培养物中多巴胺能神经元的数量。1周龄的培养物比3周龄的培养物对MPP(+)更敏感。暴露2天并恢复15天后，多巴胺耗竭的半数有效浓度（EC(50)）分别为0.6和7微摩尔。GDNF预处理和后处理对于获得针对MPP(+)毒性的最大保护都很重要。在1周龄培养物（5微摩尔MPP(+)，2天）中，GDNF提供了强大的神经保护作用，多巴胺含量达到对照水平，酪氨酸羟化酶（TH）(+)细胞数量高达对照的80%，但在3周龄培养物（10微摩尔MPP(+)，2天）中，GDNF的保护潜力明显降低。MPP(+)暴露后需要较长的恢复期来区分可逆或不可逆的毒性和/或营养作用。
