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

Summary

We present an in vitro model to assess olfactory ensheathing glia (OEG) neuroregenerative capacity, after neural injury. It is based on a coculture of axotomized adult retinal ganglion neurons (RGN) on OEG monolayers and subsequent study of axonal regeneration, by analyzing RGN axonal and somatodendritic markers.

Abstract

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory bulb. Throughout adult life, they are key for axonal growing of newly generated olfactory neurons, from the lamina propria to the ONL. Due to their pro-regenerative properties, these cells have been used to foster axonal regeneration in spinal cord or optic nerve injury models.

We present an in vitro model to assay and measure OEG neuroregenerative capacity after neural injury. In this model, reversibly immortalized human OEG (ihOEG) is cultured as a monolayer, retinas are extracted from adult rats and retinal ganglion neurons (RGN) are cocultured onto the OEG monolayer. After 96 h, axonal and somatodendritic markers in RGNs are analyzed by immunofluorescence and the number of RGNs with axon and the mean axonal length/neuron are quantified.

This protocol has the advantage over other in vitro assays that rely on embryonic or postnatal neurons, that it evaluates OEG neuroregenerative properties in adult tissue. Also, it is not only useful for assessing the neuroregenerative potential of ihOEG but can be extended to different sources of OEG or other glial cells.

Introduction

Adult central nervous system (CNS) neurons have limited regenerative capacity after injury or disease. A common strategy to promote CNS regeneration is transplantation, at the injury site, of cell types that induce axonal growth such as stem cells, Schwann cells, astrocytes or olfactory ensheathing glia (OEG) cells^1,2,3,4,5.

OEG derives from the neural crest⁶ and locates in the olfactory mucosa and in the olfactory bulb. In the adult, olfactory se....

Protocol

NOTE: Animal experimentation was approved by national and institutional bioethics committees.

1. ihOEG (Ts12 and Ts14) culture

NOTE: This procedure is done under sterile conditions in a tissue culture biosafety cabinet.

1. Prepare 50 mL ME10 OEG culture medium as provided in Table 1.
2. Prepare 5 mL of DMEM/F12-FBS, as provided in Table 1, in a 15 mL conical tube.
3. Warm both media at 37 °C in a clean water bath, for 15 min.
4. Thaw Ts12 and Ts14 cells vials at 37 °C in a clean water bath.
5. Resuspend and add cells to the DMEM/F12-....

Results

In this protocol, we present an in vitro model to assay OEG neuroregenerative capacity after neuronal injury. As shown in Figure 1, the OEG source is a reversible immortalized human OEG clonal cell line -Ts14 and Ts12-, which derives from primary cultures, prepared from olfactory bulbs obtained in autopsies^15,17,18. Retinal tissue is extracted from adult rats, digested, and retinal ganglion neurons .......

Discussion

OEG transplantation at CNS injury sites is considered a promising therapy for CNS injury due to its constitutive pro-neuroregenerative properties^7,8,9. However, depending on the tissue source—olfactory mucosa (OM-OEG) versus olfactory bulb (OB-OEG)—or the age of the donor, considerable variation exists in such capacity^26,31,33

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
antibody 514	Reference 34		Rabbit polyclonal antiserum, which recognizes MAP2A and B.
antibody SMI-31	BioLegend	801601	Monoclonal antibody against MAP1B and NF-H proteins
anti-mouse Alexa Fluor 488 antibody	ThermoFisher	A-21202
anti-rabbit Alexa Fluor 594 antibody	ThermoFisher	A-21207
B-27 Supplement	Gibco	17504044
D,L-2-amino-5-phosphonovaleric acid	Sigma	283967	NMDA receptor inhibitor
DAPI	Sigma	D9542	Nuclei fluorescent stain
DMEM-F12	Gibco	11320033	Cell culture medium
FBS	Gibco	11573397	Fetal bovine serum
FBS-Hyclone	Fisher Scientific	16291082	Fetal bovine serum
Fluoromount	Southern Biotech	0100-01	Mounting medium
ImageJ	National Institutes of Health (NIH-USA)		Image software
L-Glutamine	Lonza	BE17-605F
Neurobasal Medium	Gibco	21103049	Neuronal cells culture medium
Papain Dissociation System	Worthington Biochemical Corporation	LK003150	For use in neural cell isolation
PBS	Home made
PBS-EDTA	Lonza	H3BE02-017F
Penicillin/Streptomycin/Amphotericin B	Lonza	17-745E	Bacteriostatic and bactericidal
Pituitary extract	Gibco	13028014	Bovine pituitary extract
Poly -L- lysine (PLL)	Sigma	A-003-M