Study Glial Cell Heterogeneity Influence on Axon Growth Using a New Coculture Method

Summary

In this protocol, we described a new method to study the influence of glial cell heterogeneity on axon growth with an in vitro co-culture system. Rat cortical glial cells were cultured to confluence and cocultured with highly purified rat dorsal root ganglia neurons. Different glial cell influence on neurons adhesion and axon growth was compared directly in the same culture. This method provides a new way to directly study the glial cell heterogeneity influence on neuron adhesion and axon growth.

Abstract

In the central nervous system of all mammals, severed axons after injury are unable to regenerate to their original targets and functional recovery is very poor ¹. The failure of axon regeneration is a combined result of several factors including the hostile glial cell environment, inhibitory myelin related molecules and decreased intrinsic neuron regenerative capacity ². Astrocytes are the most predominant glial cell type in central nervous system and play important role in axon functions under physiology and pathology conditions ³. Contrast to the homologous oligodendrocytes, astrocytes are a heterogeneous cell population composed by different astrocyte subpopulations with diverse morphologies and gene expression ⁴. The functional significance of this heterogeneity, such as their influences on axon growth, is largely unknown.

To study the glial cell, especially the function of astrocyte heterogeneity in neuron behavior, we established a new method by co-culturing high purified dorsal root ganglia neurons with glial cells obtained from the rat cortex. By this technique, we were able to directly compare neuron adhesion and axon growth on different astrocytes subpopulations under the same condition.

In this report, we give the detailed protocol of this method for astrocytes isolation and culture, dorsal root ganglia neurons isolation and purification, and the co-culture of DRG neurons with astrocytes. This method could also be extended to other brain regions to study cellular or regional specific interaction between neurons and glial cells.

Protocol

1. Glia Cell Culture

Glial cells can be cultured from different regions of central nervous system. The whole process is shown in process figure.

Day 1 Coating culture plate and coverslips

1. Dry sterilized glass microscope round coverslips in an autoclave.
2. Plate the sterilized coverslips into sterilized 24-well culture plate.
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Discussion

This experiment protocol was designed to reach two goals to study glial cells, especially astrocyte heterogeneity's influence on neuron adhesion and neurite growth. The first goal was to maintain astrocyte heterogeneity as much as possible, in this experiment, the confluent glial cell culture enriched astrocytes was mixed primary culture without any chemical treatment and digestion propagation, which may further damage the cell and induce injury response of astrocytes. The final astrocyte purity is more than 90% GFAP pos.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM(high glucose)	Invitrogen	10313-039
L15 medium	Invitrogen	11415-114
FBS	Invitrogen	10437-077
Neurobasal Medium	Invitrogen	21103-049
poly-lysine	Sigma-Aldrich	P4832	culture grade
NGF(2.5S)	Invitrogen	13257-019
B27 supplyment	Invitrogen	17504-044
0.25% trypsin-EDTA	Invitrogen	25200-056
FUDR	Sigma-Aldrich	F0503
neurofilament antibody	Abcam	ab24575