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1Department of Neurosurgery, Cedars Sinai Medical Center, UCLA, 2Basic Medicine School, Fourth Military Medical University, 3Department of Neurology, David Geffen School of Medicine, UCLA, 4Aerospace Medicine School, Fourth Military Medical Univeristy
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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.
Xu, H., Gou, L., Dong, H. Study Glial Cell Heterogeneity Influence on Axon Growth Using a New Coculture Method. J. Vis. Exp. (43), e2111, doi:10.3791/2111 (2010).
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.
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
Day 2 Isolating cortex and glial cell culture
2. Dorsal Root Ganglia Neurons Isolation, Culture and Purification
Day 1 Prepare Culture Material
Day 2 Isolate DRGs from embryos
Day 3 Purify DRG Neurons
3. Coculture DRG neurons with glial cells
4. Representative Results
Figure 1. Morphology of confluent glial cells. Cortical glial cells were plated on polylysine coated coverslip and cultured for 20 days, note the different growth pattern of glial cells, cells on the left side arranged in a radiated way.
Figure 2. Confluent glial cells labeled by Glial fibrillary acidic protein (GFAP) antibody. Note the radiated arrangement of GFAP (red) positive cells on the right side.
Figure 3. Dorsal root ganglia neurons grew in vitro without FUDR treatment. The contaminating cells formed DRG neurons' background.
Figure 4. Dorsal root ganglia neurons grew in vitro after FUDR treatment. The background contaminating cells had been eliminated completely.
Figure 5. Dorsal root ganglia neurons grown on glial cells. Neurons adhesion and neurite growth were inhibited on the radiated arranged cells and limited on the right side glial cells.
Figure 6. Dorsal root ganglia neurons growing on glial cells labeled by neurofilament antibody. The neurite(green) were inhibited on the radiated arranged left side glial cells and limited on the right side, all the glial cells were labeled by GFAP antibody(red).
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 positive, with contamination fibroblasts less than 1% as verified by fibrobasts FN. The low seeding density at beginning made cells undergo several rounds of division before confluence. Under phase contrast microscope, the glial cell heterogeneity was observed by the different substructure formed by different morphological astrocytes. The second goal was to get high purified DRG neurons and coculture them with glial cells to study the heterogeneity influence on neuron behavior. Contaminating cells in dorsal root ganglia such as fibroblasts and schwann cells could apparently influence or change the axon growth in culture conditions 5, 6, to avoid this unwanted influence, DRG neurons were cultured in serum free medium and treated by FUDR for 72h to kill all the other cell types. After this treatment, DRG neurons could be purified as high as 99%. It was more efficient than traditional method which needs to treat the neurons culture several times 7. Single DRG neuron adhesion and axon growth could be easily monitored and tracked under phase contrast microscope by time lapse recording and imaging analysis method, their interaction with glial cells could be analyzed in details by immunocytochemistry and electronic microscope technique.
It was noted that DRG neurons could not be maintained in vitro for more than 4 weeks, their survival had no sign of reduction but they could not adhere to the substrate any longer, usually they would detach and roll up.
DRG neurons could be maintained on astroglial cells for a long time (more than 2 months). This long term coculture between high purified DRG neurons and astrocytes make the tracking of single neurons and axon growth on different type of astrocytes more easily, it also avoid the uncontrolled influence from other contamination cells in dorsal root ganglia. During the whole culture process, all cells should be handled carefully and all the cells should always be immersed in culture medium. To get high purity DRG neurons, a critical point is the duration of FUDR treatment, more than 72h treatment will significantly compromise cellular viability and short time treatment may not kill all the contamination cells. In the optimal condition, we can obtain large amount of high purity DRG neurons within 1 week. Using this coculture system, we found that, in contrast to commonly held beliefs, heterogeneous astrocytes had different influences on neurons adhesion and axon growth, and subpopulation astrocytes showed strong inhibition to both neuron adhesion and axon growth. Besides the interaction between astrocyte and axon growth, this method could be used in on other studies such as myelin formation, neuronal development changes in intrinsic axon growth capacity, genes and signal pathways related to axon growth. It can also be used in mice and other animals' central nervous system regions to study the interaction between neurons and glial cells.
No conflicts of interest declared.
This study was supported by FMMU new finding foundation and partially NIH funding.
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