Neuron-Macrophage Co-cultures to Activate Macrophages Secreting Molecular Factors with Neurite Outgrowth Activity

Hyeok Jun Yun; Eun-Hye Kim; Byung Gon Kim

doi:10.3791/56920

Neuron-Macrophage Co-cultures to Activate Macrophages Secreting Molecular Factors with Neurite Ou...

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JoVE Journal Neuroscience

Neuron-Macrophage Co-cultures to Activate Macrophages Secreting Molecular Factors with Neurite Outgrowth Activity

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08:52 min

March 30, 2018

DOI: 10.3791/56920-v

Hyeok Jun Yun^1,2, Eun-Hye Kim^1,2, Byung Gon Kim^1,2,3

¹Department of Brain Science,Ajou University School of Medicine, ²Neuroscience Graduate Program, Department of Biomedical Sciences,Ajou University School of Medicine, ³Department of Neurology,Ajou University School of Medicine

Overview

This study presents a protocol to stimulate cultured macrophages, enhancing their capacity to release factors that promote neurite outgrowth from neurons. The use of cyclic AMP (cAMP) in neuron-macrophage co-cultures induces macrophages to produce conditioned medium with strong neurite outgrowth activity, providing insights into the interplay between these cell types in regeneration.

Key Study Components

Area of Science

Neuroscience
Cell biology
Regenerative medicine

Background

Macrophages can influence neuron repair and growth.
Cyclic AMP is used to induce a pro-regenerative phenotype in macrophages.
Stimulating macrophages through cAMP improves axonal growth potential.

Purpose of Study

To develop a method for macrophages to secrete factors that enhance neurite growth.
To investigate interactions between neurons and macrophages in regeneration.
To validate the effect of conditioned medium on neurite outgrowth.

Methods Used

Cell culture methods were employed, specifically using neuron-macrophage co-cultures.
The study focuses on dissociated dorsal root ganglion (DRG) neurons.
CRITICAL STEPS include the preparation of coated plates, isolation of DRG, and the addition of cAMP.
The conditioned medium was collected and filtered for use in neurite outgrowth assays.

Main Results

Macrophage-conditioned medium treated with DB cyclic AMP led to significant neurite outgrowth.
Control conditioned medium did not induce neurite growth, highlighting the specific enhancement provided by cAMP.
The findings suggest that macrophage activation facilitates effective neuronal regeneration.

Conclusions

This study demonstrates a robust method for enhancing neurite outgrowth via macrophage activation.
The approach provides a valuable tool for exploring neuronal regeneration mechanisms.
The findings have implications for therapeutic strategies targeting nerve injury and neurodegenerative conditions.

Frequently Asked Questions

What are the advantages of using cAMP in this protocol?

Cyclic AMP is a physiologically relevant molecule that effectively stimulates macrophages to adopt a pro-regenerative phenotype, enhancing their ability to support neurite growth.

How are the primary macropages prepared for co-culture?

Primary peritoneal macrophages are isolated from adult mice through a method that involves injecting sterile PBS into the peritoneal cavity and collecting the lavage fluid.

What outcomes can be expected from this study?

The main outcome is the evaluation of neurite outgrowth in response to macrophage-conditioned medium, indicating the effectiveness of the macrophage stimulation protocol.

How is neuronal culture established in this study?

Dorsal root ganglion (DRG) neurons are isolated and plated onto poly-D lysine and laminin-coated plates to promote adherence and growth.

What limitations should be considered when interpreting the results?

While the study shows significant effects on neurite outgrowth, the results may vary based on cell type, experimental conditions, and tissue source.

The current protocol presents experimental procedures to stimulate cultured macrophages to be endowed with capacity to release molecular factors that promote neurite outgrowth. Treatment of cAMP to the neuron-macrophage co-cultures induces the macrophages to produce conditioned medium that possesses strong neurite outgrowth activity.

The overall goal of this procedure is to generate macrophages that can secrete molecular factors promoting neurite growth. This method can help answer key questions on how neurons and macrophages interact with each other to activate the pro-regenerative macrophagicphenotype to promote excellent regeneration. The main advantage of this technique is that we use Cyclic AMP, an adenosine serum molecule, which is much more physiologic enzymesone used in previous studies to stimulate macrophages with the pro-regenerative phenotype.

We first had the idea for this method when we found that macrophage activation is essential in the enhancement of axion growth capacity and those can produce excellent neurons following precaution in permanent lobe injury. Before setting up the culture, precode a six well plate with poly-D Lycine and laminin. Next, incubate the six well plate with 0.01 poly-D Lycine at 37 degrees Celsius for two hours or at four degrees Celsius overnight.

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