Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration

Our protocol is a simple and reproducible model to assess olfactory ensheathing capacity to foster adult axonal regeneration This is a quantitative technique that allows the evaluation of olfactory ensheathing glia regenerative capacity that can also be extended to other glial cell types. This technique is the first step to study olfactory ensheathing glia as a candidate for cell therapy in nervous system injuries before translation to in vivo or clinical studies. Demonstrating the procedure will be Maria Portela, a PhD student from our laboratory.

To begin, prepare all the required media as described in the text manuscript. After sacrificing the rat, place its head in a 100 milliliter Petri dish and spray it with 70%ethanol before placing it in a laminar flow hood. Cut the rat's whiskers with scissors so they do not interfere with the eye manipulation.

Grip the optic nerve with forceps to pull out the eyeball enough to be able to make an incision across the eye with a scalpel. Remove the lens and vitreous humor and pull out the retina, keeping the remaining layers of the eye inside. Place the retina in a previously prepared P60 cell culture dish with five milliliters of cold EBSS, then transfer it to a P60 cell culture dish with reconstituted papain plus 50 microliters of APV and 250 microliters of DNAs plus five microliters of APV.

Cut the retina into small pieces with a scalpel. Transfer the pieces to a 15 milliliter plastic tube and incubate them for 30 minutes in a humidified incubator at 37 degrees Celsius under 5%carbon dioxide, agitating every 10 minutes. Dissociate cell clumps by pipetting up and down with a glass pasture pipette.

Then centrifuge the cell suspension at 200 times G for five minutes. Discard supernatant and re-suspend the cell pellet in albumin OVO mucoid protease inhibitor with 150 microliters of DNAs and 30 microliters of APV. Carefully add the cell suspension on five milliliters of albumin OVO mucoid protease inhibitor and repeat the centrifugation.

While centrifuging, completely remove the EMI 10 medium from an OEG 24 well plate and replace it with 500 microliters of NBB 27 medium per well. Discard the supernatant and re-suspend the cells in two milliliters of NB B27 medium. Plate 100 microliters of retinal cell suspension into each well of the M 24 plate onto PLL-treated or OEG monolayer cover slips.

Maintain cultures at 37 degrees Celsius with 5%carbon dioxide for 96 hours in NB B27 medium. After 96 hours, fix the cells for 10 minutes by adding the same volume of 4%PFA in PBS to the culture medium. Discard the fixing solution and wash the cells three times with PBS for five minutes per wash, then block with PBS TS for 30 to 40 minutes.

Prepare the primary antibodies in PBS TS buffer as described in the text manuscript and add them to the cocultures. Incubate the cells overnight at four degrees Celsius. On the next day, discard the antibodies and wash the cover slips three times with PBS for five minutes per wash.

Add the corresponding fluorescent secondary antibodies and incubate the cover slips for one hour at room temperature in the dark. Then wash the cover slips three times with PBS for five minutes per wash in the dark. Mount the cover slips with mounting medium and keep them at four degrees Celsius.

Use a 40 times objective of an epi fluorescence microscope to quantify axonal regeneration. Quantify the percentage of neurons with axons relative to the total population of retinal ganglion neurons. Use the neuron J plugin in image J to quantify the axonal regeneration index or mean Ono length, which is the sum of the lengths of all identified axons divided by the total number of counted neurons, whether they presented in axon or not.

After opening the image, click on analyze and set scale according to microscope settings, working in pixels per micrometer. Select add tracings to begin axon tracking then track the axons from the soma to the end. Click on measure tracings, display tracing measurements, and run.

Neuroregenerative capacity of olfactory ensheathing glia after neuronal injury was investigated using a reversible immortalized human OEG clonal cell line. TS14 OEG identity was assessed by immunostaining within sheath and glioma markers, such as S 100 beta and vimentin. GFAP expression was also analyzed to discard astrocyte contamination.

In the axonal regeneration assay, TS14 regenerative capacity was compared to TS12 in RGN OEG cocultures using PLL substrate as a negative control. Representative images show a lack of capacity of RGN to regenerate their axons over PLL or TS12 cells. Well TS14 stimulates the outgrowth of axons in RGN.

Both the percentage of cells with axons, as well as the average length of the regenerated axons were significantly higher in neurons cocultured on TS14 monolayers compared to neurons plated on either TS12 cells or PLL. This protocol can be used to elucidate the molecular mechanisms responsible of the neuroregenerative capacities of olfactory ensheathing glia.