An in vitro assay to study induction of the regenerative state in sensory neurons.
An in vitro assay to study induction of the regenerative state in sensory neurons.
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DOI：
10.1016/j.expneurol.2014.10.012
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发表时间：
2015-01
影响因子：
5.3
通讯作者：
DiAntonio A
中科院分区：
文献类型：
--
作者：
Frey E;Valakh V;Karney-Grobe S;Shi Y;Milbrandt J;DiAntonio A
损伤后，周围神经元激活促再生程序，促进轴突再生。虽然许多再生相关基因已被确定，但损伤激活该程序的机制尚不清楚。此外，确定诱导促再生状态的药理学方法可能会导致修复受损神经系统的新疗法。因此，我们开发了一种体外实验来研究损伤或药物治疗后促再生状态的诱导。首先，我们利用了从背根神经节分离和培养感觉神经元激活促再生程序的观察结果。我们发现，在解剖后的第一个小时内，培养的神经元激活转录因子并上调与促再生程序共同的再生相关基因。在一个类似于预处理的范例中，被分离损伤的神经元在从动物身上移除后12小时内复制时，显示出增强的神经突生长。此外，促再生状态的刺激改善了抑制底物上的生长，并需要DLK/JNK信号，这两者都是体内促再生反应的标志。最后，我们修改了这个实验，以确定新的方法来激活促再生状态，以模仿预处理效果。我们报道，在培养几天后，神经元下调了许多损伤的分子标志，并且在复制后不再显示增强的神经突生长。因此，这些神经元在功能上是naïve，并且是识别诱导促再生状态的方法的有用工具。我们发现，在这种模式下，损伤和用forskolin预处理都能重新激活促再生状态。因此，该试验可用于鉴定在无损伤情况下诱导促再生状态的药理学试剂。
After injury, peripheral neurons activate a pro-regenerative program that facilitates axon regeneration. While many regeneration-associated genes have been identified, the mechanism by which injury activates this program is less well understood. Furthermore, identifying pharmacological methods to induce a pro-regenerative state could lead to novel treatments to repair the injured nervous system. Therefore, we have developed an in vitro assay to study induction of the pro-regenerative state following injury or pharmacological treatment. First, we took advantage of the observation that dissociating and culturing sensory neurons from dorsal root ganglia activates a pro-regenerative program. We show that cultured neurons activate transcription factors and upregulate regeneration-associated genes common to the pro-regenerative program within the first hours after dissection. In a paradigm similar to pre-conditioning, neurons injured by dissociation display enhanced neurite outgrowth when replated as early as 12 hours after being removed from the animal. Furthermore, stimulation of the pro-regenerative state improves growth on inhibitory substrates and requires DLK/JNK signaling, both hallmarks of the pro-regeneration response in vivo. Finally, we modified this assay in order to identify new methods to activate the pro-regenerative state in an effort to mimic the pre-conditioning effect. We report that after several days in culture, neurons down-regulate many molecular hallmarks of injury and no longer display enhanced neurite outgrowth after replating. Hence, these neurons are functionally naïve and are a useful tool for identifying methods to induce the pro-regenerative state. We show that both injury and pre-treatment with forskolin reactivate the pro-regenerative state in this paradigm. Hence, this assay is useful for identifying pharmacological agents that induce the pro-regenerative state in the absence of injury.
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DOI：
10.1073/pnas.251194298
发表时间：
2001-11-20
影响因子：
11.1
作者：
Bennett, BL;Sasaki, DT;Anderson, DW
通讯作者：
Anderson, DW
影响因子：
16.2
作者：
Kadoya, Ken;Tsukada, Shingo;Lu, Paul;Coppola, Giovanni;Geschwind, Dan;Filbin, Marie T.;Blesch, Armin;Tuszynski, Mark H.
通讯作者：
Tuszynski, Mark H.
影响因子：
3.5
作者：
Lindwall, C;Dahlin, L;Kanje, M
通讯作者：
Kanje, M
影响因子：
4.7
作者：
Gao, Ying;Deng, Kangwen;Walsh, Frank S.
通讯作者：
Walsh, Frank S.
影响因子：
5.3
作者：
Christie, Kimberly J.;Webber, Christine A.;Zochodne, Douglas W.
通讯作者：
Zochodne, Douglas W.