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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 sensory neurons die regularly as the result of environmental exposure and they are replaced by newly differentiated neurons. OEG surrounds and guides these new olfactory axons to enter the olfactory bulb and to establish new synapses with their targets in the CNS⁷. Due to these physiological attributes, OEG has been used in models of CNS injury such as spinal cord or optic nerve injury and its neuroregenerative and neuroprotective properties become proven^8,9,10,11. Several factors have been identified as responsible of the pro-regenerative characteristics of these cells, including extracellular matrix proteases production or secretion of neurotrophic and axonal growth factors^12,13,14.

Given the technical limitations to expand primary OEG cells, we previously established and characterized reversible immortalized human OEG (ihOEG) clonal lines, which provide an unlimited supply of homogeneous OEG. These ihOEG cells derive from primary cultures, prepared from olfactory bulbs obtained in autopsies. They were immortalized by transduction of the telomerase catalytic subunit (TERT) and the oncogene Bmi-1 and modified with the SV40 virus large T antigen^15,16,17,18. Two of these ihOEG cell lines are Ts14, which maintains the regenerative capacity of the original cultures and Ts12, a low regenerative line that is used as a low regeneration control in these experiments¹⁸.

To assess OEG capacity to foster axonal regeneration after neural injury, several in vitro models have been implemented. In these models, OEG is applied to cultures of different neuronal origin and neurite formation and elongation—in response to glial coculture—are assayed. Examples of such neuronal sources are neonatal rat cortical neurons¹⁹, scratch wounds performed on rat embryonic neurons from cortical tissue²⁰, rat retinal explants²¹, rat hypothalamic or hippocampal postnatal neurons^22,23, postnatal rat dorsal root ganglion neurons²⁴, postnatal mouse corticospinal tract neurons²⁵, human NT2 neurons²⁶, or postnatal cerebral cortical neurons on reactive astrocyte scar-like cultures²⁷.

In these models, however, the regeneration assay relies on embryonic or postnatal neurons, which have an intrinsic plasticity that is absent in injured adult neurons. To overcome this drawback, we present a model of adult axonal regeneration in cocultures of OEG lines with adult retinal ganglion neurons (RGNs), based on the one originally developed by Wigley et al.^28,29,30,31 and modified and used by our group^{12,13,14,15,16,17,18,32,33}. Briefly, retinal tissue is extracted from adult rats and digested with papain. Retinal cell suspension is then plated on either polylysine-treated coverslips or onto Ts14 and Ts12 monolayers. Cultures are maintained for 96 h before they are fixed and then immunofluorescence for axonal (MAP1B and NF-H proteins)³⁴ and somatodendritic (MAP2A and B)³⁵ markers is performed. Axonal regeneration is quantified as a percentage of neurons with axon, with respect to the total population of RGNs and axonal regeneration index is calculated as the mean axonal length per neuron. This protocol is not only useful for assessing the neuroregenerative potential of ihOEG but can be extended to different sources of OEG or other glial cells.

Protokół

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-FBS culture medium prepared in step 2.
6. Centrifuge for 5 min at 200 x g.
7. Aspire the supernatant.
8. Add 500 µL of ME10 medium and resuspend the pellet.
9. Prepare a p60 cell culture dish with 3 mL of ME10 and add the cellular suspension, dropwise.
10. Move to distribute the cells uniformly across the plate.
11. Culture cells at 37 °C in 5% CO₂.
    NOTE: After reaching confluence, at least another passage must be done to optimize cells for coculture. 90% confluence is needed before seeding them on the coverslips for coculture. A confluent p-60 has a mean cell number of 7 x 10⁵ for Ts14 and 2.5 x 10⁶ for Ts12 cell lines. Ts12 and Ts14 cell lines should be passaged every 2–3 days.

2. Preparation of ihOEG (Ts12 and Ts14) for the assay

NOTE: This step must be done 24 h before RGN dissection and coculture.

1. Treat 12 mm Ø coverslips with 10 µg/mL poly-L-lysine (PLL) for 1 h.
   NOTE: The coverslips can be left overnight in PLL solution.
2. Wash the coverslips with 1x phosphate buffer saline (PBS), three times.
3. Detach Ts12 and Ts14 ihOEG cells from p60 cell culture dish.
   1. Add 4 mL of DMEM/F12-FBS culture medium (Table 1) to a 15 mL conical tube. Warm at 37 °C in a clean water bath.
   2. Remove the medium from plates and wash cells with 1 mL of 1x PBS-EDTA, once.
   3. Add 1 mL of trypsin-EDTA to the OEG cells and incubate for 3–5 min at 37 °C, 5% CO₂.
   4. Collect cells with a p1000 pipette and transfer them to medium prepared in step 3.1.
   5. Centrifuge for 5 min at 200 x g.
   6. Aspire the supernatant.
   7. Add 1 mL of ME10 medium and resuspend the pellet.
   8. Count the cell number in a hemocytometer.
4. Seed 80,000 Ts14 cells or 100,000 Ts12 cells/well onto the coverslips in 24-well plates in 500 µL of ME10 medium.
5. Culture cells at 37 °C in 5% CO₂, for 24 h.

3. Retinal tissue dissection

NOTE: 2-month old male Wistar rats are used as RGN source. Two retinas (one rat) for 20 wells of a 24-well cell dish. Autoclave surgical material before use. Papain dissociation kit is commercially purchased (Table of Materials). Follow the provider´s instructions for reconstitution. Reconstitute D,L-2-amino-5-phosphonovaleric acid (APV) in 5 mM stock and prepare the aliquots.

1. On the day of the assay, prepare the following media.
   1. Prepare a p60 cell culture dish with 5 mL of cold EBSS (vial 1 of the papain dissociation kit).
   2. Prepare a p60 cell culture dish with reconstituted vial 2 (papain) of the papain dissociation kit plus 50 µL of APV.  Then, add 250 µL ofreconstituted vial 3 (DNase plus 5 µL of APV).
   3. In a sterile tube mix 2.7 mL of vial 1 with 300 µL of vial 4 (albumin-ovomucoid protease inhibitor). Add 150 µL of vial 3 (DNase) plus 30 µL of APV.
   4. Prepare 20 mL of Neurobasal-B27 medium (NB-B27) as provided in Table 1.
2. Sacrifice a rat by asphyxiation with CO₂.
3. Remove the head by decapitation with guillotine; place it in a 100 mm Petri dish and spray the head with ethanol 70% before placing it in a laminar flow hood.
4. Cut the rat´s whiskers with scissors so they do not interfere with the eye manipulation.
5. 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.
6. Remove the lens and vitreous humor and pull out the retina (orange-like tissue), while the remaining layers of the eye stay inside (including the pigment epithelial layer).
7. Place the retina in the p60 cell culture dish prepared in step 3.1.1.
8. Transfer the retina to the p60 cell culture dish prepared in step 3.1.2 and cut it with the scalpel in small pieces of an approximate size < 1 mm.
9. Transfer to a 15 mL plastic tube.
10. Incubate the tissue for 30 min, in a humidified incubator at 37 °C under 5% CO₂, with agitation every 10 min.
11. Dissociate cell clumps by pipetting up and down with a glass Pasteur pipette.
12. Centrifuge the cell suspension at 200 x g for 5 min.
13. Discard supernatant and to inactivate papain, resuspend the cell pellet in the solution prepared in step 3.1.3. (1.5 mL for 2 eyes).
14. Carefully pipet this cell suspension into 5 mL of reconstituted vial 4.
15. Centrifuge at 200 x g for 5 min.
16. While centrifuging, completely remove the ME-10 medium from the OEG 24 well cell plate (previously prepared in step 2) and replace it with 500 µL of NB-B27 medium per well.
17. Discard the supernatant and resuspend the cells in 2 mL of NB-B27 medium.
18. Plate 100 µL of retinal cell suspension, per well of the m24 plate, onto PLL-treated or OEG monolayers-coverslips.
19. Maintain cultures at 37 °C with 5% CO₂ for 96 h in NB-B27 medium.

4. Immunostaining

1. After 96 h, fix the cells for 10 min by adding the same volume of 4% paraformaldehyde (PFA) in 1x PBS to the culture medium (600 µL) (PFA final concentration 2%).
2. Remove the media and PFA from the 24-multiwell plate and once again add 500 µL of 4% paraformaldehyde (PFA) in 1x PBS. Incubate for 10 min.
3. Discard the fixer and wash 3 times with 1x PBS for 5 min.
4. Block with 0.1% Triton X-100/1% FBS in PBS (PBS-TS) for 30–40 min.
5. Prepare the primary antibodies in PBS-TS buffer as follows: SMI31 (against MAP1B and NF-H proteins) monoclonal antibody (1:500). 514 (recognizes MAP2A and B proteins) rabbit polyclonal antiserum (1:400).
6. Add primary antibodies to cocultures and incubate overnight at 4 °C.
7. Next day, discard the antibodies and wash the coverslips with 1x PBS, 3 times, for 5 min.
8. Prepare the secondary antibodies in PBS-TS buffer as follows: For SMI-31, anti-mouse Alexa Fluor 488 (1:500). For 514, anti-rabbit Alexa-594 (1:500).
9. Incubate cells with the corresponding fluorescent secondary antibodies for 1 h, at RT, in the dark.
10. Wash the coverslips with 1x PBS, 3 times, for 5 min, in the dark.
11. Finally, mount coverslips with mounting medium (Table of Materials) and keep at 4 °C.
    NOTE: Whenever necessary, fluorescent nuclei staining with DAPI (4,6-diamidino-2-phenylindole) may be performed. Before mounting, incubate the cells for 10 min in the dark with DAPI (10 µg/mL in 1x PBS). Wash the coverslips 3 times with 1x PBS and finally, mount the coverslips with the mounting medium.

5. Axonal regeneration quantification

NOTE: Samples are quantified under the 40x objective of an epifluorescence microscope. A minimum of 30 pictures should be taken on random fields, with at least 200 neurons, to be quantified for each treatment. Each experiment should be repeated a minimum of three times.

1. Quantify the percentage of neurons with axon (SMI31 positive neurite) relative to the total population of RGNs (identified with MAP2A/B 514 positive immunostaining of neuronal body and dendrites).
2. Quantify the axonal regeneration index or mean axonal length (µm/neuron). This parameter is defined as the sum of the lengths (in µm) of all identified axons, divided by the total number of counted neurons, whether they presented an axon or not. Axonal length is determined using the plugin NeuronJ of the image software ImageJ (NIH-USA).
3. Calculate the mean, standard deviation, and statistical significance using the appropriate software.

Wyniki

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 ...

Dyskusje

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

Materiały

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