Abstract
Macrophages are known to play beneficial roles in axon regeneration after nerve injury. To develop an in vitro model in which injury signals can elicit pro-regenerative macrophage activation, we established co-cultures consisting of adult dorsal root ganglia sensory neurons and peritoneal macrophages and added cAMP analogue dibutyryl cAMP. The conditioned medium collected from the co-cultures exhibited robust neurite outgrowth activities. The neurite outgrowth activities were almost completely abrogated by addition of minocycline, a macrophage deactivator, indicating that factors responsible for neurite outgrowth are produced by activated macrophages.
Background
CNS neurons of adult mammals do not spontaneously regenerate axons after injury. Preconditioning peripheral nerve injury allows the dorsal root ganglia (DRG) sensory axons to regenerate central branches by promoting expression of regeneration-associated genes. We have previously showed that activated macrophages in the DRG following preconditioning injury critically contribute to enhancement intrinsic regeneration capacity of the DRG sensory neuron (Kwon et al., 2013). To identify molecular factors involved in the activation of macrophages following nerve injury, we have developed an in vitro model in which neuron-macrophages interactions are elicited by cAMP, a well-known reagent to enhance regenerative capacity of neurons. Compared to the previous model to activate macrophages using zymosan, our model utilizes a more physiologic stimulus resembling molecular events in the preconditioning peripheral injury model.
Materials and Reagents
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Data analysis
Notes
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Acknowledgments
This protocol is supported by a grant NRF-2015R1A2A1A01003410 from the Ministry of Science, ICT and Future Planning, Republic of Korea.
References
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