Name: coculture of axotomized rat retinal ganglion neurons with olfactory ensheathing glia, as an in vitro model of adult axonal regeneration
Uploaded: 2020-11-02T15:00:00.000+00:00
Duration: 7 min 57 s
Description: Watch this Scientific Journal Video about Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration at JoVE.com

This work was financially supported by project SAF2017-82736-C2-1-R from Ministerio de Ciencia e Innovaci&#243;n to MTM-F and by Fundaci&#243;n Universidad Francisco de Vitoria to JS.

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.
<ol>
	<li>Prepare 50 mL ME10 OEG culture medium as provided in Table 1.</li>
	<li>Prepare 5 mL of DMEM/F12-FBS, as provided in Table 1, in a 15 mL conical tube.</li>
	<li>Warm both media at 37 &#176;C in a clean water bath, for 15 min.</li>
	<li>Thaw Ts12 and Ts14 cells vials at 37 &#176;C in a clean water bath.</li>
	<li>Resuspend and add cells to the DMEM/F12-FBS culture medium prepared in step 2.</li>
	<li>Centrifuge for 5 min at 200 x g.</li>
	<li>Aspire the supernatant.</li>
	<li>Add 500 &#181;L of ME10 medium and resuspend the pellet.</li>
	<li>Prepare a p60 cell culture dish with 3 mL of ME10 and add the cellular suspension, dropwise.</li>
	<li>Move to distribute the cells uniformly across the plate.</li>
	<li>Culture cells at 37 &#176;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&#8211;3 days.</li>
</ol>
2. Preparation of ihOEG (Ts12 and Ts14) for the assay
NOTE: This step must be done 24 h before RGN dissection and coculture.
<ol>
	<li>Treat 12 mm &#216; coverslips with 10 &#181;g/mL poly-L-lysine (PLL) for 1 h. 
	NOTE: The coverslips can be left overnight in PLL solution.</li>
	<li>Wash the coverslips with 1x phosphate buffer saline (PBS), three times.</li>
	<li>Detach Ts12 and Ts14 ihOEG cells from p60 cell culture dish.
	<ol>
		<li>Add 4 mL of DMEM/F12-FBS culture medium (Table 1) to a 15 mL conical tube. Warm at 37 &#176;C in a clean water bath.</li>
		<li>Remove the medium from plates and wash cells with 1 mL of 1x PBS-EDTA, once.</li>
		<li>Add 1 mL of trypsin-EDTA to the OEG cells and incubate for 3&#8211;5 min at 37 &#176;C, 5% CO2.</li>
		<li>Collect cells with a p1000 pipette and transfer them to medium prepared in step 3.1.</li>
		<li>Centrifuge for 5 min at 200 x g.</li>
		<li>Aspire the supernatant.</li>
		<li>Add 1 mL of ME10 medium and resuspend the pellet.</li>
		<li>Count the cell number in a hemocytometer.</li>
	</ol>
	</li>
	<li>Seed 80,000 Ts14 cells or 100,000 Ts12 cells/well onto the coverslips in 24-well plates in 500 &#181;L of ME10 medium.</li>
	<li>Culture cells at 37 &#176;C in 5% CO2, for 24 h.</li>
</ol>
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&#180;s instructions for reconstitution. Reconstitute D,L-2-amino-5-phosphonovaleric acid (APV) in 5 mM stock and prepare the aliquots.
<ol>
	<li>On the day of the assay, prepare the following media.
	<ol>
		<li>Prepare a p60 cell culture dish with 5 mL of cold EBSS (vial 1 of the papain dissociation kit).</li>
		<li>Prepare a p60 cell culture dish with reconstituted vial 2 (papain) of the papain dissociation kit plus 50 &#181;L of APV.&#160; Then, add 250 &#181;L ofreconstituted vial 3 (DNase plus 5 &#181;L of APV).</li>
		<li>In a sterile tube mix 2.7 mL of vial 1 with 300 &#181;L of vial 4 (albumin-ovomucoid protease inhibitor). Add 150 &#181;L of vial 3 (DNase) plus 30 &#181;L of APV.</li>
		<li>Prepare 20 mL of Neurobasal-B27 medium (NB-B27) as provided in Table 1.</li>
	</ol>
	</li>
	<li>Sacrifice a rat by asphyxiation with CO2.</li>
	<li>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.</li>
	<li>Cut the rat&#180;s whiskers with scissors so they do not interfere with the eye manipulation.</li>
	<li>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.</li>
	<li>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).</li>
	<li>Place the retina in the p60 cell culture dish prepared in step 3.1.1.</li>
	<li>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 &#60; 1 mm.</li>
	<li>Transfer to a 15 mL plastic tube.</li>
	<li>Incubate the tissue for 30 min, in a humidified incubator at 37 &#176;C under 5% CO2, with agitation every 10 min.</li>
	<li>Dissociate cell clumps by pipetting up and down with a glass Pasteur pipette.</li>
	<li>Centrifuge the cell suspension at 200 x g for 5 min.</li>
	<li>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).</li>
	<li>Carefully pipet this cell suspension into 5 mL of reconstituted vial 4.</li>
	<li>Centrifuge at 200 x g for 5 min.</li>
	<li>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 &#181;L of NB-B27 medium per well.</li>
	<li>Discard the supernatant and resuspend the cells in 2 mL of NB-B27 medium.</li>
	<li>Plate 100 &#181;L of retinal cell suspension, per well of the m24 plate, onto PLL-treated or OEG monolayers-coverslips.</li>
	<li>Maintain cultures at 37 &#176;C with 5% CO2 for 96 h in NB-B27 medium.</li>
</ol>
4. Immunostaining
<ol>
	<li>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 &#181;L) (PFA final concentration 2%).</li>
	<li>Remove the media and PFA from the 24-multiwell plate and once again add 500 &#181;L of 4% paraformaldehyde (PFA) in 1x PBS. Incubate for 10 min.</li>
	<li>Discard the fixer and wash 3 times with 1x PBS for 5 min.</li>
	<li>Block with 0.1% Triton X-100/1% FBS in PBS (PBS-TS) for 30&#8211;40 min.</li>
	<li>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).</li>
	<li>Add primary antibodies to cocultures and incubate overnight at 4 &#176;C.</li>
	<li>Next day, discard the antibodies and wash the coverslips with 1x PBS, 3 times, for 5 min.</li>
	<li>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).</li>
	<li>Incubate cells with the corresponding fluorescent secondary antibodies for 1 h, at RT, in the dark.</li>
	<li>Wash the coverslips with 1x PBS, 3 times, for 5 min, in the dark.</li>
	<li>Finally, mount coverslips with mounting medium (Table of Materials) and keep at 4 &#176;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 &#181;g/mL in 1x PBS). Wash the coverslips 3 times with 1x PBS and finally, mount the coverslips with the mounting medium.</li>
</ol>
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.
<ol>
	<li>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).</li>
	<li>Quantify the axonal regeneration index or mean axonal length (&#181;m/neuron). This parameter is defined as the sum of the lengths (in &#181;m) 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).</li>
	<li>Calculate the mean, standard deviation, and statistical significance using the appropriate software.</li>
</ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+distinguish+phosphorylated+and+nonphosphorylated+forms+of+neurofilaments+in+situ.">Monoclonal antibodies distinguish phosphorylated and nonphosphorylated forms of neurofilaments in situ.</a> Proceedings of the National Academy of Sciences of the United States of America. 80 (19), 6126-6130 (1983).</li><li>S&#225;nchez Martin, C., D&#237;az-Nido, J., Avila, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+a+site-specific+phosphorylation+of+the+microtubule-associated+protein+2+during+the+development+of+cultured+neurons.">Regulation of a site-specific phosphorylation of the microtubule-associated protein 2 during the development of cultured neurons.</a> Neuroscience. 87 (4), 861-870 (1998).</li><li>Reshamwala, R., Shah, M., Belt, L., Ekberg, J. A. K., St. John, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliable+cell+purification+and+determination+of+cell+purity:+crucial+aspects+of+olfactory+ensheathing+cell+transplantation+for+spinal+cord+repair.">Reliable cell purification and determination of cell purity: crucial aspects of olfactory ensheathing cell transplantation for spinal cord repair.</a> Neural Regeneration Research. 15 (11), 2016-2026 (2020).</li></ol>

The authors have nothing to disclose.

OEG transplantation at CNS injury sites is considered a promising therapy for CNS injury due to its constitutive pro-neuroregenerative properties7,8,9. However, depending on the tissue source&#8212;olfactory mucosa (OM-OEG) versus olfactory bulb (OB-OEG)&#8212;or the age of the donor, considerable variation exists in such capacity26,31,33,36. Therefore, it is of importance to have an easy and reproducible in vitro model to assay the neuroregenerative capacity of a given OEG sample, before initiating in vivo studies. In this protocol, adult rats&#8217; axotomized RGN are cocultured onto a monolayer of the OEG to assay. Subsequent analysis of RGN axonal and somatodendritic markers by immunofluorescence is performed to assess RGN axonal regeneration.
An initial difficulty of the assay is the source of OEG. In this work, we use reversible immortalized human OEG (ihOEG) clonal lines, previously established and characterized by our group15,16,17,18, which provide an unlimited supply of homogeneous OEG. 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 Nevertheless, although technical limitations exist to expand human primary OEG cells, they can also be obtained from nasal endoscopic biopsies&#8212;OM&#8212;or, in case of OB-OEG, from cadaver donors.
Preparation of monolayer OEG cultures is a crucial procedure, as too many cells could cause the coculture to detach from the plate. Therefore, prior to OEG preparation for the assay, it is recommended that the user determines the optimal number of cells to be plated, depending on their size and division rate.
Another critical issue is the retinal tissue dissociation, after retina dissection. It is necessary to break up the tissue fragments, following incubation in the dissociation mix. If done too vigorously, the cells will be destroyed, but tissue fragments will be left intact if done too weakly. In order to obtain a homogeneous cell suspension, we suggest filling and emptying a Pasteur pipette 10&#8211;15 times, with a tip of intermediate diameter, while avoiding bubbling. Pasteur pipettes with wide tips can be narrowed using a Bunsen burner.
To assess the capacity of different glial populations to foster adult neurons&#8217; axonal regeneration, we have determined that 96 h is the time interval that best suits the aim because: 1) it is the longest time to maintain the culture alive without disturbing the OEG monolayer; and 2) it is the time needed for neurons to grow axons long enough to reveal differences between the regenerative capacities of different OEG populations or other non-regenerative cells (i.e., fibroblasts12,13,14,15,16,17,18,32,33). It would certainly be interesting to determine the time course of the regeneration process, as it could provide information about the differential regenerative properties of different glial populations, at shorter times of the co-culture. In our hands, for regenerative glia, the time course between 72&#8211;96 h is quite similar for all the cell lines, although axons are shorter at 72 h (unpublished data). Also, 96 h of co-culture, permits to study OEG-dependent mechanisms of adult axonal regeneration12,14.
During axonal regeneration quantification, it is important to take a minimum of 30 pictures at 400 augments (40x objective), at different random areas of the coverslip, but following the complete axons of the photographed neurons. Therefore, the experimenter must take serial pictures in the chosen areas to measure the real axonal lengths.
Other in vitro approaches have also been developed to evaluate OEG regenerative functions. In these models, OEG is applied to cultures of different neuronal origin and, in response to glial coculture, neurite formation and elongation are assayed19,20,21,22,23,24,25,26,27. However, the regeneration assay relies on embryonic or postnatal neurons, which have an intrinsic plasticity absent from injured adult neurons. This model consisting of adult axonal regeneration in cocultures of OEG lines with adult retinal ganglion neurons (RGNs) overcomes this drawback. In addition, we are dissecting adult retinas, and because we cut optic nerve and axons retract in the process of dissection, we obtain neuronal bodies clean of myelin, to perform the coculture. This is the difference with other parts of the adult CNS, where myelin can hinder very much with the dissection to obtain clean neurons for the coculture.
Based on the one originally developed by Wigley et al.28,29,30,31, we highlight the following improvements in the protocol. First, the use of neurobasal medium supplemented with B27 as OEG-RGN coculture medium, which allows growth of neuronal cells and positively affects the reproducibility of the experiment. Second, we characterize and quantify axonal regeneration by using a specific marker of the axonal compartment; and third, we use an additional direct parameter, the mean axonal length/neuron, that assesses the axonal growth regenerative potential of OEG.
In summary, we consider that this is a simple, reproducible, time saving, and medium-cost assay, not only useful for assessing the neuroregenerative potential of ihOEG, but also because it can be extended to different sources of OEG or other glial cells. Moreover, it could be used as a valuable proof of concept of the neuroregenerative potential of an OEG or glial sample, before translation to in vivo or clinical studies.

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) cells1,2,3,4,5.
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 proven8,9,10,11. 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 factors12,13,14.
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 antigen15,16,17,18. 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&#8212;in response to glial coculture&#8212;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 neurons22,23, 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 group12,13,14,15,16,17,18,32,33. 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.

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

coculture of axotomized rat retinal ganglion neurons with olfactory ensheathing glia, as an in vitro model of adult axonal regeneration

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.

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 autopsies15,17,18. Retinal tissue is extracted from adult rats, digested, and retinal ganglion neurons (RGN) suspension is plated on either PLL-treated coverslips or onto ihOEG monolayers, Ts14 or Ts12. Cultures are maintained for 96 h before they are fixed. Axonal and somatodendritic markers are analyzed by immunofluorescence and axonal regeneration is quantified.
Ts14 OEG identity is assessed by immunostaining with markers described to be expressed in ensheathing glia (Figure 2), such as S100 &#946; (Figure 2A) and vimentin (Figure 2B); GFAP expression was also analyzed to discard astrocyte contamination (Figure 2C). As shown, Ts14 expressed S100 &#946; and vimentin but not GFAP.
In the axonal regeneration assay, Ts14 regenerative capacity is compared to Ts12 in RGN-OEG cocultures, using PLL substrate as a negative control (Figure 3). 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 (Figure 3D,E). Representative images show a lack of capacity of RGN to regenerate their axons over PLL or Ts12 cells (Figure 3A,B), while Ts14 stimulates the outgrowth of axons in RGN (3C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61863/61863fig01v2.jpg" /> 
Figure 1: Diagram of rat retinal ganglion neurons with olfactory ensheathing glia cells coculture, as a model of adult axonal regeneration.&#160;Immortalized human OEG (ihOEG) clonal cell lines -Ts12 and Ts14- derived from primary cultures from olfactory bulbs. Retinal ganglion neurons from adult rats are plated on either PLL-treated coverslips (negative control) or onto Ts14 or Ts12 monolayers. Cultures are maintained for 96 h before they are fixed and axonal and somatodendritic markers are analyzed by immunofluorescence. Percentage of neurons with axon and mean axonal length/neuron are quantified to assay RGN axonal regeneration. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61863/61863fig01v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61863/61863fig02v2.jpg" /> 
Figure 2: Identity of ihOEG cell line Ts14.&#160;Immunofluorescence images of Ts14 in culture, labeled with anti-S100 &#946; (panel A, green) and vimentin (panel B, red). GFAP expression (panel C, red) was also analyzed to discard astrocyte contamination. Nuclei are stained with DAPI (blue). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61863/61863fig02v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61863/61863fig03.jpg" /> 
Figure 3: Assay for axonal regeneration in cocultures of OEG lines with adult retinal ganglion neurons (RGNs).&#160;(A&#8211;C) Immunofluorescence images showing somatodendritic labelling with 514 antibody, which recognizes microtubule-associated protein MAP2A and B, in red, and with axon-specific SMI31 antibody in green, against MAP1B and NF-H proteins. Green arrows indicate RGN axons (SMI31-positive: green) and yellow arrows indicate neuronal bodies and dendrites (514 positive: red and yellow). (D,E) Graphs show mean and standard deviation of the percentage of neurons exhibiting axons and the axonal regeneration index, a parameter reflecting the mean axonal length (&#181;m) of axons per neuron. A minimum of 30 pictures (40x) were taken on random fields and quantified for each cell sample. Experiments were performed in triplicate, from three different rats (N = 3), retinal tissue pooled from both eyes, with duplicates for each experimental condition (each glia population tested). Asterisks indicate the statistical significance: *p &#60; 0.05, **p &#60; 0.01, ***p &#60; 0.001, NS: non significance (ANOVA and post hoc Tukey test comparisons between parameters quantified for Ts14 vs Ts12, Ts14 vs PLL, and Ts12 vs PLL). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61863/61863fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration at JoVE.com

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

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.

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 ...

coculture-axotomized-rat-retinal-ganglion-neurons-with-olfactory

Nanyang Technological University

Research

JoVE Journal

Neuroscience

3.8K Views.  Universidad Francisco de Vitoria. We presenteren een in vitro model om olfactorisch verwarmend glia (OEG) neuroregeneratief vermogen te beoordelen, na neuraal letsel. Het is gebaseerd op een cocultuur van axotomized volwassen retinale ganglion neuronen (RGN) op OEG monolagen en daaropvolgende studie van axonale regeneratie, door het analyseren van RGN axonale en somatodendritische markers.

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

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

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.

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.

In the central nervous system of all mammals, severed axons after injury are unable to regenerate to their original targets and functional recovery is ...

Methods for Experimental Manipulations after Optic Nerve Transection in the Mammalian CNS

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals through the vitreous fluid ensures that they act upon the entire RGC population. Viral vectors, plasmids or short interfering RNAs (siRNAs) can also be delivered to the vitreous chamber in order to infect or transfect retinal cells 5-12. The high tropism of Adeno-Associated Virus (AAV) vectors is beneficial to target RGCs, with an infection rate approaching 90% of cells near the injection site 6, 7, 13-15. Moreover, RGCs can be selectively transfected by applying siRNAs, plasmids, or viral vectors to the cut end of the optic nerve 16-19 or injecting vectors into their target the superior colliculus 10. This allows researchers to study apoptotic mechanisms in the injured neuronal population without confounding effects on other bystander neurons or surrounding glia. RGC apoptosis has a characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a window for experimental manipulations directed against pathways involved in apoptosis. Manipulations that directly target RGCs from the transected optic nerve stump are performed at the time of axotomy, immediately after cutting the nerve. In contrast, when substances are delivered via an intraocular route, they can be injected prior to surgery or within the first 3 days after surgery, preceding the initiation of apoptosis in axotomized RGCs. In the present article, we demonstrate several methods for experimental manipulations after optic nerve transection.

Optic Nerve transection is a widely used model of adult CNS injury. This model is ideal for performing a number of experimental manipulations that target the retina globally or directly target the injured neuronal population of retinal ganglion cells.

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be ...

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved4. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth5-7.
Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons9.

An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. Studying neurons in vivo can be challenging due to their complexity, their varied and dynamic environments, and technical limitations. For these reasons, studying neurons in vitro can prove beneficial to unravel the complex mysteries of neurons. The well-defined nature of cell culture models provides detailed control over environmental conditions and variables. Here we describe how to isolate, dissociate, and culture primary neurons from chick embryos. This technique is rapid, inexpensive, and generates robustly growing sensory neurons. The procedure consistently produces cultures that are highly enriched for neurons and has very few non-neuronal cells (less than 5%). Primary neurons do not adhere well to untreated glass or tissue culture plastic, therefore detailed procedures to create two distinct, well-defined laminin-containing substrata for neuronal plating are described. Cultured neurons are highly amenable to multiple cellular and molecular techniques, including co-immunoprecipitation, live cell imagining, RNAi, and immunocytochemistry. Procedures for double immunocytochemistry on these cultured neurons have been optimized and described here.

Cell culture models provide detailed control over environmental conditions and thus provide a powerful platform to elucidate numerous aspects of neuronal cell biology. We describe a rapid, inexpensive, and reliable method to isolate, dissociate, and culture sensory neurons from chick embryos. Details of substrata preparation and immunocytochemistry are also provided.

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. ...

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various intrinsic and extrinsic inhibitory factors involved in the restriction of axon regeneration have been identified. However, simply removing these inhibitory cues is insufficient for successful regeneration, indicating the existence of additional regulatory machinery. Drosophila melanogaster, the fruit fly, shares evolutionarily conserved genes and signaling pathways with vertebrates, including humans. Combining the powerful genetic toolbox of flies with two-photon laser axotomy/dendriotomy, we describe here the Drosophila sensory neuron &#8211; dendritic arborization (da) neuron injury model as a platform for systematically screening for novel regeneration regulators. Briefly, this paradigm includes a) the preparation of larvae, b) lesion induction to dendrite(s) or axon(s) using a two-photon laser, c) live confocal imaging post-injury and d) data analysis. Our model enables highly reproducible injury of single labeled neurons, axons, and dendrites of well-defined neuronal subtypes, in both the peripheral and central nervous system.

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

Here, we present a protocol using the Drosophila sensory neuron - dendritic arborization (da) neuron injury model, which combines in vivo live imaging, two-photon laser axotomy/dendriotomy, and the powerful fly genetic toolbox, as a platform for screening potential promoters and inhibitors of neuroregeneration.

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various ...

Medicine

Sheath-Preserving Optic Nerve Transection in Rats to Assess Axon Regeneration and Interventions Targeting the Retinal Ganglion Cell Axon

Retinal ganglion cell (RGC) axons converge at the optic nerve head to convey visual information from the retina to the brain. Pathologies such as glaucoma, trauma, and ischemic optic neuropathies injure RGC axons, disrupt transmission of visual stimuli, and cause vision loss. Animal models simulating RGC axon injury include optic nerve crush and transection paradigms. Each of these models has inherent advantages and disadvantages. An optic nerve crush is generally less severe than a transection and can be used to assay axon regeneration across the lesion site. However, differences in crush force and duration can affect tissue responses, resulting in variable reproducibility and lesion completeness. With optic nerve transection, there is a severe and reproducible injury that completely lesions all axons. However, transecting the optic nerve dramatically alters the blood brain barrier by violating the optic nerve sheath, exposing the optic nerve to the peripheral environment. Moreover, regeneration beyond a transection site cannot be assessed without reapposing the cut nerve ends. Furthermore, distinct degenerative changes and cellular pathways are activated by either a crush or transection injury.
The method described here incorporates the advantages of both optic nerve crush and transection models while mitigating the disadvantages. Hydrostatic pressure delivered into the optic nerve by microinjection completely transects the optic nerve while maintaining the integrity of the optic nerve sheath. The transected optic nerve ends are reapposed to allow for axon regeneration assays. A potential limitation of this method is the inability to visualize the complete transection, a potential source of variability. However, visual confirmation that the visible portion of the optic nerve has been transected is indicative of a complete optic nerve transection with 90-95% success. This method could be applied to assess axon regeneration promoting strategies in a transection model or investigate interventions that target the axonal compartments.

This protocol describes an optic nerve transection that preserves the optic nerve sheath in rats. Hydrostatic pressure from microinjections into the optic nerve generate a complete transection, allowing for sutureless reapposition of the transected optic nerve ends and direct targeting of the axonal compartment in a transection model.

Retinal ganglion cell (RGC) axons converge at the optic nerve head to convey visual information from the retina to the brain. Pathologies such as ...

In Vitro Myelination of Peripheral Axons in a Coculture of Rat Dorsal Root Ganglion Explants and Schwann Cells

The process of myelination is essential to enable rapid and sufficient signal transduction in the nervous system. In the peripheral nervous system, neurons and Schwann cells engage in a complex interaction to control the myelination of axons. Disturbances of this interaction and breakdown of the myelin sheath are hallmarks of inflammatory neuropathies and occur secondarily in neurodegenerative disorders. Here, we present a coculture model of dorsal root ganglion explants and Schwann cells, which develops a robust myelination of peripheral axons to investigate the process of myelination in the peripheral nervous system, study axon-Schwann cell interactions, and evaluate the potential effects of therapeutic agents on each cell type separately. Methodologically, dorsal root ganglions of embryonic rats (E13.5) were harvested, dissociated from their surrounding tissue, and cultured as whole explants for 3 days. Schwann cells were isolated from 3-week-old adult rats, and sciatic nerves were enzymatically digested. The resulting Schwann cells were purified by magnetic-activated cell sorting and cultured under neuregulin and forskolin-enriched conditions. After 3 days of dorsal root ganglion explant culture, 30,000 Schwann cells were added to one dorsal root ganglion explant in a medium containing ascorbic acid. The first signs of myelination were detected on day 10 of coculture, through scattered signals for myelin basic protein in immunocytochemical staining. From day 14 onward, myelin sheaths were formed and propagated along the axons. Myelination can be quantified by myelin basic protein staining as a ratio of the myelination area and axon area, to account for the differences in axonal density. This model provides experimental opportunities to study various aspects of peripheral myelination in vitro, which is crucial for understanding the pathology of and possible treatment opportunities for demyelination and neurodegeneration in inflammatory and neurodegenerative diseases of the peripheral nervous system.

In the coculture system of dorsal root ganglia and Schwann cells, myelination of the peripheral nervous system can be studied. This model provides experimental opportunities to observe and quantify peripheral myelination and to study the effects of compounds of interest on the myelin sheath.

The process of myelination is essential to enable rapid and sufficient signal transduction in the nervous system. In the peripheral nervous system, ...

Coculturing of Retinal Ganglion Neurons with Olfactory Ensheathing Glia

Source: Portela-Lomba, M., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/61863">Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration.</a> J. Vis. Exp. (2020)
This video demonstrates the procedure to isolate retinal ganglion neurons (RGNs) from a rat&#39;s retina and coculture them with olfactory ensheathing glia (OEG) cells. This method allows for studying the neuro-regenerative potential of OEGs after neural injury.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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Neuronal Culture Techniques

Co-culture and Interaction Studies

Co-culturing of a Dorsal Root Ganglion Explant with Schwann Cells for Neuron Myelination

Source: Blusch, A. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/64768">In Vitro Myelination of Peripheral Axons in a Coculture of Rat Dorsal Root Ganglion Explants and Schwann Cells.</a> J. Vis. Exp. (2023)
This video demonstrates the coculturing model of a dorsal root ganglion explant and Schwann cells. In coculture conditions, ascorbic acid promotes the differentiation of Schwann cells into myelinating forms. Myelinating Schwann cells wrap the axons multiple times to create a myelin sheath, which is essential for nerve function.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. 
1. Schwann cell culture

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

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