Name: dorsal root ganglia neurons and differentiated adipose-derived stem cells: an in vitro co-culture model to study peripheral nerve regeneration
Uploaded: 2015-02-26T14:00:00
Duration: 9 min 17 s
Description: Watch this Scientific Journal Video about Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration at JoVE.com

This work is supported by the National Institute for Health Research, Academy of Medical Sciences and the British Society for Surgery of the Hand. We also gratefully acknowledge the continuing supply of GGF-2 from Acorda Therapeutics, USA. The authors would finally like to acknowledge Prof. Giorgio Terenghi for his valuable support and guidance in our group over the past years that led to the development and optimization of this protocol.

NOTE: All the experiments involving animals were carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986.
1. Experimental Set-up
<ol>
	<li>Prior to start tissue and cell harvest, check that all the tools are sterile. Whether necessary, autoclave a pair of sharp surgical scissors, a very fine forceps and a fine standard forceps. Also sterilize each substrate before cell seeding using UV sterilization, ethanol exposure or steam sterilization as appropriate.</li>
	<li>Preparation of media for stem cell harvest and differentiation
	<ol>
		<li>Prepare the stem cell growth medium, containing Minimum Essential Medium (&#945;-MEM) supplemented with 10 % fetal bovine serum (FBS), 200 mM L-glutamine, and 1 % penicillin-streptomycin (PS).</li>
		<li>Prepare a 10 mM stock solution of forskolin by dissolving 10 mg of forskolin in 2.436 ml of sterile dimethyl sulfoxyde. Use at final concentration of 14 &#956;M.</li>
		<li>Prepare a 35 mg/ml stock solution of retinoic acid by dissolving 50 mg in 1.43 ml of sterile dimethyl sulfoxyde. Use at final concentration of 350 ng/ml.</li>
		<li>Prepare platelet-derived growth factor (PDGF) stocks (100 &#956;g/ml) by dissolving 10 &#956;g of lyophilized powder in 100 &#956;l of distilled sterile water. Use at final concentration of 5 ng/ml.</li>
		<li>Prepare basic fibroblast growth factor (bFGF) stocks (100 &#956;g/ml) by dissolving 50 &#956;g of lyophilized powder in 500 &#956;l of distilled sterile water. Use at final concentration of 10 ng/ml.</li>
		<li>Prepare the stem cell differentiation medium, containing stem cell growth medium supplemented with 14 &#181;M forskolin, 126 ng/ml glial growth factor-2 (GGF-2), 5 ng/mL platelet-derived growth factor (PDGF), and 10 ng/ml basic fibroblast growth factor (bFGF).</li>
	</ol>
	</li>
	<li>Preparation of media and stock solutions for neuron harvest and dissociation
	<ol>
		<li>Prepare Bottenstein and Sato&#8217;s (BS) medium28, by adding 1 % PS and 1 % N2 supplement to Ham&#8217;s F12 medium. Calculate the final volume needed as 500 &#181;l per well (if using a 24-well plate).</li>
		<li>Prepare collagenase IV stocks in Ham&#8217;s F12 medium at the concentration of 1.25 % wt/v. Filter-sterilize the solution and store at -20 &#176;C as 200 &#181;l aliquots.</li>
		<li>Prepare bovine pancreatic trypsin stocks in Ham&#8217;s F12 medium at the concentration of 2.5 % wt/v, filter-sterilize and store at -20 &#176;C as 200 &#181;l aliquots.</li>
		<li>Prepare the nerve growth factor (NGF) stock solution at the concentration of 5 &#181;g/mL in a filter-sterilized 1 mg/ml fatty acid-free bovine serum albumin (BSA) solution in F12 medium and store at -20 C as 200 &#181;l aliquots. Do not filter the NGF after reconstitution.</li>
	</ol>
	</li>
	<li>In case no surface modification has been performed, coat coverslips/plates with poly-D-lysine (0.1 mg/ml for 15 min at RT) and/or laminin (2-10 &#181;g/cm2 for 2 hr at 37 &#176;C) to support neuron attachment and neurite outgrowth as appropriate.</li>
	<li>Always warm up media in a water bath at 37 &#176;C before using.</li>
</ol>
2. Adipose-derived Stem Cell (ASC) Harvest and Differentiation into a SC Phenotype
<ol>
	<li>ASC harvest from visceral and inguinal fat of adult male Sprague-Dawley rats
	<ol>
		<li>Prior to start, prepare a tube with 10-15 ml of Hanks&#8217; Balanced Saline Solution (HBSS) supplemented with 1% v/v of PS solution and store on ice until fat harvesting.</li>
		<li>Terminate the rat by cervical dislocation and decapitation. Shave the rat and make an incision through the abdominal skin, exposing the internal organs and avoiding bleeding. Remove the visceral fat encasing the stomach and intestines (usually characterized by a fatty yellow consistency) and the inguinal fat surrounding the testicles from an adult male Sprague-Dawley rat. Transfer the fat in the tube containing HBSS on ice.</li>
		<li>In a biological safety cabinet (class II) finely chop the fat using a pair of scissors and a sterile razor blade until fine consistency is reached and transfer it into a tube containing 15 ml of 0.2 % wt/v collagenase type I solution freshly prepared and filter-sterilized on the day.</li>
		<li>Transfer the tube in a water bath at 37 &#176;C and leave the fat tissue to digest in presence of the enzyme for 30 min-1 hr under continuous agitation. Monitor the digestion closely and stop before the tissue is fully dissociated, this will improve cell viability and cell yield. Filter theun-dissociated tissue through a 100 &#181;m cell strainer. 
		NOTE: Good tissue digestion will result in homogenous consistency of the fat, visible by eye when gently swirling the tube, acquiring a beige appearance.</li>
		<li>Neutralize the enzyme by adding 15 ml of stem cell growth medium containing fetal bovine serum at 37 &#176;C and centrifuge the solution at 160 g for 10 min in order to collect the stromal vascular fraction, including the stem cells, at the bottom of the tube.</li>
		<li>At this stage, the pellet can be resuspended in 1 ml of red blood cells lysis buffer to remove blood cell contamination. After resuspension and pipetting for 1 min, add 10 ml of fresh stem cell growth medium and centrifuge at 160 g for 10 min.</li>
		<li>Carefully aspirate the supernatant from the tube, taking care of the deposited cell pellet at the bottom. Resuspend the cells in 10 ml of stem cell growth medium, transfer them into a 75 cm2 flasks and incubate at 37 &#176;C, 5 % CO2. Maintain the cells at sub-confluent levels until passage 1-2, changing medium every 3 days.</li>
	</ol>
	</li>
	<li>ASC differentiation to a SC phenotype
	<ol>
		<li>At passage 1-2, remove the stem cell growth medium from the 75 cm2 flask and replace it with 10 ml of fresh medium containing 1 mM &#946;-mercaptoethanol freshly prepared and filter-sterilized on the day. Incubate the cells at 37 &#176;C, 5 % CO2 for 24 hr. At this stage, it is important that the cells are seeded at low density (30 %) before starting the differentiation.</li>
		<li>Wash the cells carefully with HBSS, aspirate and replace it with 10 ml of medium containing 350 ng/ml retinoic acid. Incubate the cells at 37 &#176;C, 5 % CO2 for 72 hr. Try to minimize exposure of cell medium to light.</li>
		<li>After 3 days, wash the cells carefully with HBSS, aspirate and replace it with 10 ml of stem cell differentiation medium (see step 1.2.6). Maintain the cells at sub-confluent levels, changing medium every 3 days. 
		NOTE: Following 2 weeks of incubation, ASC are differentiated into SC-like ASC, expressing their characteristic phenotype (as demonstrated by Kingham et al.5). They can then be used up until the 10th passage without noticeable changes in behavior29 .</li>
	</ol>
	</li>
</ol>
3. Harvest and Dissociation of Dorsal Root Ganglia (DRG) Neurons
<ol>
	<li>Terminate the rat by cervical dislocation and decapitation. Shave the rat and lift the skin to expose the spinal column. Using sharp scissors, excide the spinal column taking extra care of the confined organs and blood vessels. Transfer the spinal column in a biological safety cabinet (class II) using a Petri dish and remove any dorsal part.</li>
	<li>Divide the spinal column in half along the longitudinal axis using sterile and sharp surgical scissors to expose the cord tissue. At this point, it is helpful to cut the spinal column in two smaller segments below the level of the rib cage, to make it easier to handle during the DRG neuron harvest. Using fine forceps, gently remove all the cord tissue, paying attention to not pull and remove the DRG roots. This way the DRG and roots will be exposed within the vertebral canals, still encased in the column. Observe DRG as white filaments coming out directly from the canals.</li>
	<li>Pull out the entire DRG root (not just the DRG) from the vertebral canals by using very fine forceps, going deep into the vertebral canals and paying attention not to damage the roots of the ganglia. Transfer the DRG into a small petri dish (60 mm2) containing 3-4 ml of Ham&#8217;s F12 medium supplemented with 1% PS. If using different animals, use separate dishes.</li>
	<li>Under a dissecting microscope, clean the DRG of any excess of nerve roots surrounding the ganglia using sterile forceps and a scalpel to reduce glial cell contamination. Transfer the DRG into a small petri dish (35 mm2) with 1.8 ml of fresh F12 medium.</li>
	<li>Add 200 &#181;l of 1.25 % wt/v collagenase type IV stock solution (final concentration of 0.125%) and incubate the DRG at 37 &#176;C, 5 % CO2 for 1 hr. Carefully aspirate the medium with a glass pipette, paying attention not to aspirate or damage the DRG. Add fresh F12 medium supplemented with 0.125 % wt/v collagenase type IV enzyme and incubate for 1 hr as previously described.</li>
	<li>Aspirate the medium and gently wash the DRG with F12 medium. Add then 1.8 ml of F12 medium and 200 &#181;l of trypsin (final concentration of 0.25 % wt/v) and incubate at 37 &#176;C, 5 % CO2 for 30 min.</li>
	<li>Remove trypsin and add 1 ml of F12 medium supplemented with 500 &#181;l of FBS to arrest the enzymatic reaction. Aspirate the medium and gently wash the DRG with F12 medium for three times to remove traces of serum.</li>
	<li>Add 2 ml of fresh F12 medium and carefully transfer the DRG with the medium into a 15 ml tube using a glass pipette. Gently dissociate the DRG neurons by pipetting up and down (about 8-10 times) with the glass pipette (plastic tips can be used as alternative).</li>
	<li>Allow the pellet to settle at the bottom of the tube and collect the medium in a new tube. Add 2 ml of fresh F12 medium to the tube containing the pellet and repeat the mechanical dissociation with the glass pipette. Repeat this step until the suspension becomes homogeneous (about 3-4 times) and collect all the dissociated DRG in the new tube. This method reduces the stress deriving from the mechanical dissociation and improves the viability of the neurons.</li>
	<li>Filter the resulting homogenized suspension into a new 15 ml tube using a 100 &#181;m cell strainer to remove un-dissociated neurons and other debris. At this stage, it might be convenient to first filter the cell suspension into a 50 ml tube and then transfer the solution into the smaller tube using a glass pipette. Centrifuge the suspension at 110 g for 5 min.</li>
	<li>Prepare a 15 % bovine serum albumin (BSA) by adding 500 &#181;l of a 30 % BSA solution to 500 &#181;l of F12 medium. Slowly pipette the solution down the wall of a 15 ml tube to create a gradual protein trail. At this stage, it is helpful to hold the tube at a 45&#176; angle and slowly release the BSA using the numbers on the falcon tube as a reference for the forming &#8220;track&#8221;.</li>
	<li>Aspirate the supernatant from step 3.10 leaving 500 &#181;l at the bottom of the tube and resuspend the cell pellet in the same medium. Slowly pipette the suspension along the protein trail previously prepared in step 3.11 (use the numbers on the tube as a reference) and centrifuge at 500 g for 5 min.</li>
	<li>Aspirate the supernatant and resuspend the pellet in 1 ml of modified BS medium (or a mixed medium for the SC-like ASC/DRG co-culture, as described below in step 4.5. 
	NOTE: The volume to re-suspend the DRG neurons can be made up to the actual volume required, depending on the final cell concentration desired and the number of samples to be seeded. One animal will provide enough cells for a 24-well plate experiment.</li>
	<li>Incubate the seeded samples at 37 &#176;C, 5% CO2 for 2 hr to allow cell attachment and finally add fresh BS medium supplemented with 50 ng/ml of nerve growth factor (NGF).</li>
</ol>
4. Direct Co-culture of SC-like ASC and DRG Neurons
<ol>
	<li>Maintain SC-like ASC in culture at sub-confluent levels as described in step 2.2.3. Twenty-four hr prior to DRG harvest, aspirate the stem cell differentiation medium and wash the cells with HBSS. Aspirate and incubate in 3 ml of trypsin at 37 &#176;C, 5% CO2 for 3 min.</li>
	<li>Check under a light microscope that all the cells have detached from the flask. Gently tap the flask to help detachment. Add 7 ml of medium to stop the trypsin reaction, collect the cell suspension in a 15 ml tube and centrifuge at 110 g for 5 min.</li>
	<li>Aspirate the supernatant and resuspend the cell pellet in 5 ml of stem cell differentiation medium. Count the cells using a hemocytometer and dilute the cell suspension according to the final concentration required. Ideally, seeding 20,000 SC-like ASC/cm2 would guarantee a confluent layer of cells.</li>
	<li>Seed the SC-like ASC on the substrate and incubate at 37 &#176;C, 5 % CO2 for 24 hr to allow cell attachment.</li>
	<li>After 24 hr incubation, aspirate the medium and add the DRG neurons on top of the cells as described in step 3.14 and 3.15. Change the medium to a mixed medium containing 50 % of stem cell differentiation medium and 50 % of BS medium. In addition, reducing the FBS concentration to 1-2.5 % in the final mixed medium helps avoiding the proliferation of contaminating satellite cells derived from the DRG dissociation.</li>
	<li>Incubate the co-cultured samples at 37 &#176;C, 5 % CO2 and maintain in culture for the required time for future tests.</li>
</ol>

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C., Faroni, A., Downes, S., Terenghi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiated+adipose-derived+stem+cells+act+synergistically+with+RGD-modi+fi+ed+surfaces+to+improve+neurite+outgrowth+in+a+co-culture+model.">Differentiated adipose-derived stem cells act synergistically with RGD-modi fi ed surfaces to improve neurite outgrowth in a co-culture model.</a> J Tissue Eng Regen Med. , (2013).</li><li>Summa, P. G., Kalbermatten, D., Raffoul, W., Terenghi, G., Kingham, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+Matrix+Molecules+Enhance+the+Neurotrophic+Effect+of+Schwann+Cell-Like+Differentiated.">Extracellular Matrix Molecules Enhance the Neurotrophic Effect of Schwann Cell-Like Differentiated.</a> Tissue Eng. 19 (3-4), 368-379 (2013).</li><li>Fudge, N. J., Mearow, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix-associated+gene+expression+in+adult+sensory+neuron+populations+cultured+on+a+laminin+substrate.">Extracellular matrix-associated gene expression in adult sensory neuron populations cultured on a laminin substrate.</a> BMC Neurosci. 14 (15), 1-19 (2013).</li><li>Stabenfeldt, S. E., LaPlaca, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variations+in+rigidity+and+ligand+density+influence+neuronal+response+in+methylcellulose-laminin+hydrogels.">Variations in rigidity and ligand density influence neuronal response in methylcellulose-laminin hydrogels.</a> Acta Biomater. 7 (12), 4102-4108 (2011).</li><li>Terenghi, G., Wiberg, M., Kingham, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+21:+Use+of+stem+cells+for+improving+nerve+regeneration.">Chapter 21: Use of stem cells for improving nerve regeneration.</a> Inl Revi Neurobiol. 87 (09), 393-403 (2009).</li><li>Richardson, J. a., Rementer, C. W., Bruder, J. M., Hoffman-Kim, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidance+of+dorsal+root+ganglion+neurites+and+Schwann+cells+by+isolated+Schwann+cell+topography+on+poly(dimethyl+siloxane)+conduits+and+films.">Guidance of dorsal root ganglion neurites and Schwann cells by isolated Schwann cell topography on poly(dimethyl siloxane) conduits and films.</a> J Neural Eng. 8 (4), 046015 (2011).</li><li>Seggio, a. M., Narayanaswamy, A., Roysam, B., Thompson, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-aligned+Schwann+cell+monolayers+demonstrate+an+inherent+ability+to+direct+neurite+outgrowth.">Self-aligned Schwann cell monolayers demonstrate an inherent ability to direct neurite outgrowth.</a> J Neural Eng. 7 (4), 046001 (2010).</li><li>Xu, F. J., Wang, Z. H., Yang, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+functionalization+of+polycaprolactone+films+via+surface-initiated+atom+transfer+radical+polymerization+for+covalently+coupling+cell-adhesive+biomolecules.">Surface functionalization of polycaprolactone films via surface-initiated atom transfer radical polymerization for covalently coupling cell-adhesive biomolecules.</a> Biomaterials. 31 (12), 3139-3147 (2010).</li><li>Armstrong, S. J., Wiberg, M., Terenghi, G., Kingham, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ECM+molecules+mediate+both+Schwann+cell+proliferation+and+activation+to+enhance+neurite+outgrowth.">ECM molecules mediate both Schwann cell proliferation and activation to enhance neurite outgrowth.</a> Tissue Eng. 13 (12), 2863-2870 (2007).</li></ol>

The authors confirm that there are no conflicts of interest associated with this publication.

DRG neurons are frequently used among neuronal cells for primary culture to study the regeneration of neurons after axotomy in vivo. Here an accurate protocol for DRG harvest from adult rats is presented, aimed at reducing the population of satellite cells in the surrounding environment without compromising the neuronal survival. As ASC differentiated into a SC-like phenotype are a valid alternative to SC for cell therapies, a SC-like ASC/DRG co-culture system is also described in detail.
It is widely known that laminin (or laminin-derived peptide sequences) have a beneficial effect on neuron survival and neurite formation31&#8211;33. When performing DRG neuron cultures it is therefore advised to previously coat each substrate with laminin in order to avoid any loss of functionality of the neuronal cells. The concept of laminin coatings also applies to biomaterial substrates for the design of tissue engineering constructs, such as poly--caprolactone (PCL) frequently used for the fabrication of nerve conduits30. Also, previous work demonstrated that fibrin matrices are suitable materials for neuronal cultures in three dimension11.
In addition to protein coating, co-culture models provide valuable conditions for DRG neuron survival and a suitable environment to study the interactions that occur between neurons and SC in the peripheral nervous system following injury. Cells can also be transplanted in vivo by using neural devices in order to shorten the recruitment time of autologous cells at the injury site. This is particularly important in severe injuries, which can lead to cell senescence/death and muscle atrophy. Although SC are the most important glial cells involved in the process of peripheral nerve regeneration and myelination, their limited availability and their slow proliferation rate makes them unsuitable for tissue engineering applications34. ASC are a valid alternative due to their abundance and ability to differentiate into the SC phenotype, expressing specific glial markers and showing functional similarities to native SC19. ASC are also able to produce proteins and growth factors5 that can be beneficial to the formation and extension of neurites by DRG neurons in a co-culture system. However, two different co-culture systems can be set up as function of the experimental needs. The method proposed in this paper is a revised version of a well-established protocol in our laboratory11 and it involves a direct contact between the two cell types (direct co-culture), in which the second cell type (DRG neurons) are seeded on the top of the others (SC-like ASC). This approach is based on previous findings that demonstrated the importance of the presence of a glial cell layer on the substrate when seeding neuronal cultures35&#8211;37. This effect is likely due to cell-cell interactions through DRG integrins and ECM molecules deposited from ASC and other cues on the stem cell surface. It was also observed that a reduction of protein serum in the medium reduced the proliferation of contaminating satellite cells that can derive from the dissociation of DRG neurons, without affecting ASC functions. However, satellite cells, including a small population of SC, are hard to completely eliminate from cultures of DRG neurons and few remaining cells will also be present in the co-culture system. It is therefore important to note that these small sub-populations can also participate to myelination processes during in vitro studies, recalling the autologous cells that are present in vivo after injury. The second approach (not presented here) involves the use of cell culture inserts, avoiding a direct contact between the two different cell types (indirect co-culture). However, it is not representative of the in vivo conditions during nerve regeneration (reduced ability of neuronal cells to develop long neurites), but it is used to investigate the effect of released diffusible factors in the medium by a certain cell population onto the other38.

Peripheral nerve injuries are common with approximately 9,000 cases in the UK occurring each year in a predominantly young and working population1. Despite microsurgical nerve repair techniques, normal restoration of function is unattainable with resulting impaired hand sensation, reduced motor function and frequent pain and cold intolerance2. Such injuries have a profound and permanent impact on the patient and their ability to perform activities of daily living, with less than 60 % returning to work3.
Following injury, the phenotype and the morphology of neurons and Schwann cells (SC) change in order to create a suitable environment to allow the axon sprouting. In case of transection, the nerve is divided into proximal and distal stumps; the proximal stump being the point from which the regenerative process takes place, whilst the distal stump undergoes Wallerian degeneration whereupon the SC detach from the injured axons, de-differentiate and proliferate. This is fundamental towards removing myelin debris and preparing the distal stump for nerve re-generation 4,5. Axon sprouting is supported by the production of neurotrophic factors and chemokines released by SC at the distal stump, and guided by the basal lamina left behind following Wallerian degeneration6,7. SC align alongside the regenerating axon forming the bands of B&#252;ngner, which aid the axon growth towards the target organ, reducing branching outside the endoneurial tube. Following reinnervation, SC form the new myelin sheath wrapping the regenerated axons, but sensory and motor function is only partially restored8.
Dorsal root ganglia (DRG) are structures located in the intervertebral foramina of the spinal column, containing the sensory neuronal cells innervating the peripheral organs. When dissociated, they can be used as a suitable in vitro model for the study of nerve regeneration9&#8211;11, including investigations of myelin formation. In particular, adult DRG neurons mimic the in vivo characteristics of these cells and provide a formidable tool to study new strategies for peripheral nerve repair in tissue engineering.
Co-cultures represent a dynamic system that simulates in vitro the interplay of two (or more) cell types in a particular in vivo environment. One of the advantages of these cell co-culture models is the flexibility and high control that can be exerted on the extracellular environment. DRG neurons have been used frequently in co-culture systems with SC to mimic the actual interactions that occur between the two cell types in the peripheral nervous system10,12&#8211;14. It was demonstrated that SC secrete extracellular matrix (ECM) proteins and growth factors that can remarkably improve the ability of DRG neurons to survive and sprout neurites15,16. However, SC require lengthy periods of time to proliferate and, despite the advances in cell culture technique, it is still hard to generate a suitable number of cells for tissue engineering applications. In addition, the sacrifice of a healthy nerve is necessitated to harvest autologous SC. Therefore, the difference in sourcing SC is important for both tissue engineering and in vitro testing of nerve regeneration. In this view, ASC can be considered a valuable alternative for the development of a tissue-engineered construct to be used for peripheral nerve repair17,18. Previous work demonstrated the ability of these cells to differentiate into SC-like ASC, expressing characteristic glial-markers, such as S-100, p75 and glial fibrillary acidic protein (GFAP)19, as well as the myelin protein zero (P0)20. The secretion of glial growth factors, such as brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and glial cell-derived neurotrophic factor (GDNF) was also observed21,22. Therefore, SC-like ASC can be used as promoter of peripheral nerve regeneration, as demonstrated by both in vitro and in vivo studies23&#8211;26. In addition, ASC can be harvested through minimally-invasive procedures in higher number compared to other stem cell types; the frequency of stem cells in adipose tissue is 100- to 1,000-fold higher than in bone marrow27,and they have a higher proliferation rate compared to SC and bone marrow mesenchymal stem cells.
This work aims to provide a detailed protocol to perform high efficient harvests of dissociated DRG neurons and ASC respectively, the latter being differentiated into SC-like cells. The co-culture of these two cell types will therefore provide a very practical system that can be used for future studies on the ability of DRG neurons to sprout neurites and the mechanisms of myelin formation on different scaffold for nerve tissue engineering.

<table border="0" cellpadding="0" cellspacing="0" width="618">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:116px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">100 &#181;m cell strainer</td>
			<td style="width:116px;">BD Biosciences</td>
			<td style="width:119px;">352360</td>
			<td style="width:167px;">70 &#956;m strainers (ref. 352350) can be used as alternative</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">15 mL plastic tubes</td>
			<td style="width:116px;">Sarstedt</td>
			<td style="width:119px;">62.554.002</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">50 mL plastic tubes</td>
			<td style="width:116px;">Sarstedt</td>
			<td style="width:119px;">62.547.004</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">75 cm2 flasks</td>
			<td style="width:116px;">Corning</td>
			<td style="width:119px;">BC301</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Retinoic Acid &#62;98% HPLC</td>
			<td style="width:116px;">Sigma</td>
			<td style="width:119px;">R2625</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">ARA-C supplement</td>
			<td style="width:116px;">Sigma</td>
			<td style="width:119px;">C6645</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Recombinant Human FGF-basic (154 aa)</td>
			<td style="width:116px;">Peprotech</td>
			<td style="width:119px;">100-18B</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:116px;">Sigma</td>
			<td style="width:119px;">A9205</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Collagenase type I</td>
			<td style="width:116px;">Gibco</td>
			<td style="width:119px;">17100-017</td>
			<td style="width:167px;">Note: this collagenase is only used for fat tissue digestion</td>
		</tr>
		<tr height="63">
			<td height="84" rowspan="2" style="height:84px;width:216px;">Collagenase type IV</td>
			<td rowspan="2" style="width:116px;">Worthington Biochemical</td>
			<td rowspan="2" style="width:119px;">LS004188</td>
			<td rowspan="2" style="width:167px;">Note: this collagenase is only used to dissociate DRG explants</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Foetal Bovine Serum (FBS)</td>
			<td style="width:116px;">Biosera</td>
			<td style="width:119px;">FB-1001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Forskolin&#160;</td>
			<td style="width:116px;">Sigma</td>
			<td style="width:119px;">F3917</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Glass pipettes</td>
			<td style="width:116px;">Fisher Scientific</td>
			<td style="width:119px;">FB50253</td>
			<td style="width:167px;">Sharp material to be disposed accordingly</td>
		</tr>
		<tr height="253">
			<td height="253" style="height:253px;width:216px;">Glial Growth Factor-2 (GGF-2)</td>
			<td style="width:116px;">Acorda Therapeutics</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">GGF-2 was kindly donated by Acorda Therapeutics. For a commercially available alternative, we recommend NRG1-&#946;1 (R &#38; D Systems, Abingdon) for stem cell differentiation to be used at the final concentration of 200ng/ml</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Nutrient Mix F12 HAM</td>
			<td style="width:116px;">Sigma</td>
			<td style="width:119px;">N6658</td>
			<td style="width:167px;">Warm at 37 &#176;C in a water bath unless specified</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Hank&#8217;s Balanced Salt Solution (HBSS)</td>
			<td style="width:116px;">Sigma</td>
			<td style="width:119px;">H9394</td>
			<td style="width:167px;">Warm at 37 &#176;C in a water bath unless specified</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">N-2 supplement (100x)</td>
			<td style="width:116px;">Invitrogen</td>
			<td style="width:119px;">17502</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Nerve Growth Factor 2.5s Protein, Mouse Submaxillary Glands&#160; (NGF)</td>
			<td style="width:116px;">Millipore</td>
			<td style="width:119px;">NC011</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Penicillin-Streptomycin (PS)</td>
			<td style="width:116px;">Sigma</td>
			<td style="width:119px;">P0781</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Petri dishes</td>
			<td style="width:116px;">Corning</td>
			<td style="width:119px;">430165</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Recombinant Human PDGF-AA</td>
			<td style="width:116px;">Peprotech</td>
			<td style="width:119px;">100-13A</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:216px;">Trypsin&#160;</td>
			<td style="width:116px;">Invitrogen</td>
			<td style="width:119px;">25200-056</td>
			<td style="width:167px;">Warm at 37 &#176;C in a water bath. This is used for cell detachment from tissue culture flasks</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Trypsin (2x bovine pancreatic)</td>
			<td style="width:116px;">Worthington Biochemical</td>
			<td style="width:119px;">LS003703</td>
			<td style="width:167px;">This is used for DRG dissociation</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Minimum Essential Medium Eagle (MEM)</td>
			<td style="width:116px;">Sigma</td>
			<td style="width:119px;">M8042</td>
			<td style="width:167px;">Warm at 37 &#176;C in a water bath unless specified</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">2-mercaptoethanol</td>
			<td style="width:116px;">Sigma</td>
			<td style="width:119px;">M3148</td>
			<td style="width:167px;">Prepare the solution in the biological cabinet</td>
		</tr>
	</tbody>
</table>

dorsal root ganglia neurons and differentiated adipose-derived stem cells: an in vitro co-culture model to study peripheral nerve regeneration

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.

Cultures of dissociated DRG neurons represent a suitable in vitro model for the study of nerve regeneration. However, untreated substrates do not provide a suitable environment for DRG attachment and extension of neurites. SC-like ASC are able to produce growth factors and chemokines19 which can improve the ability of DRG neurons to sprout neurites when they are released in the culture medium. The presence of SC-like ASC in the culture system can therefore play an important role in regulating the DRG functions.
This protocol (Figure 1) illustrates the procedure to perform a direct co-culture of SC-like ASC and DRG neurons, during which neuronal cells get in direct contact with previously seeded SC-like ASC. The main difficulty associated with this procedure is the high variability of the number of DRG neurons harvested from different animals. For this reason, results can be sometimes hard to compare and a particular attention should be paid during the seeding procedure in order to obtain always a comparable cell density in each experiment. The protocol has been optimized to reduce at least the number of satellite cells remaining from the DRG neuron dissociation using the BSA gradient (step 3.11). In addition, cytosine-arabinose (ARA-C) supplement can be added to the BS medium in order to further minimize the satellite cell population, as described by Kingham et al.11. However, the choice of the treatment time needs to be carefully balanced with the DRG and SC-like ASC vitality, also affected by the presence of ARA-C supplement in the culture medium. Therefore, it is very hard to accomplish a satellite cell free-system.
Figure 2 shows the importance of SC-like ASC for DRG neurite sprouting in a co-culture model using untreated and chemically-modified poly--caprolactone (PCL) films as substrates. DRG neurons were maintained in culture for 3 days in presence or absence of SC-like ASC, using a mixed solution containing 50 % of BS medium and 50 % of stem cell differentiation medium, as described in step 4.5. Following this period, cells were fixed in 4 % paraformaldehyde and stained with &#946;-tubulin III (DRG neurons) and S100 (SC-like ASC) to investigate cell morphology and to study the ability of DRG neurons to protrude neurites. No neurites were observed on untreated surfaces in absence of SC-like ASC (Figure 2A), whilst the formation of neurites was clearly improved in the co-culture system (Figure 2C). On average, the number of neurites per cell body significantly increased from 0 to 3 in presence of stem cells. Confirmation of these results was also given by scanning electron microscopy (SEM) analysis, shown in Figure 3. In particular, the SEM images show that the ability of DRG neurons to sprout neurites occurs preferentially in conjunction with SC-like ASC, as indicated by the yellow arrows in Figure 3C.
It should be pointed out that the use of laminin-modified substrates (including laminin-derived peptides) in culture systems containing DRG neurons has been frequently defined as a suitable condition for neuronal cell culture, having remarkable effects on neurite formation and extension30&#8211;32. However, the results shown in Figure 2D and Figure 3D demonstrates that the combination of chemical and biological cues can further enhance the response of DRG neurons.
<img alt="Figure 1" src="/files/ftp_upload/52543/52543fig1highres.jpg" /> 
Figure 1.&#160;Preparation of the direct DRG-SC-like ASC co-culture system. DRG neurons and ASC are derived respectively from the spinal column and the visceral and inguinal fat of adult male Sprague-Dawley rats. After the digestion of the adipose tissue through a cascade of enzymatic reactions, ASC are differentiated into SC-like cells and maintained in sub-confluent conditions until needed. SC-like ASC are pre-seeded on each substrate 24 hr prior the DRG harvest. On the day, DRG neurons are extracted and dissociated through a series of enzymatic and mechanical actions. The neurons are then seeded on the top of the previously seeded SC-like ASC and maintained in culture until assayed (mixed medium: containing 50 % of stem cell differentiation medium and 50 % of modified BS medium). <a href="https://www.jove.com/files/ftp_upload/52543/52543fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52543/52543fig2highres.jpg" /> 
Figure 2.&#160;Fluorescence images of DRG neurons in diverse culture conditions. (A) untreated PCL films; (B) RGD-modified PCL films; (C) co-cultured with SC-like ASC on untreated PCL films; (D) co-cultured with SC-like ASC on RGD-modified PCL films. Cells were maintained in culture for 3 days using a mixed solution containing 50 % of modified BS medium and 50 % of stem cell differentiation medium. After this time, cells were fixed in 4 % paraformaldehyde, permeabilized in a Triton-X solution, and non-specific binding sites were blocked with 1 % BSA. Neuronal cells were then stained against &#946;-tubulin III (FITC; green) and SC-like ASC against S-100 (AlexaFluor568; red) antibodies. Finally nuclei were stained with DAPI (blue). Images were acquired using a fluorescence microscope (Olympus BX60, Japan). (Re-print with permission from de Luca et al.30. <a href="https://www.jove.com/files/ftp_upload/52543/52543fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52543/52543fig3highres.jpg" /> 
Figure 3.&#160;SEM images of DRG neurons indiverse culture conditions. (A) untreated PCL films (B) RGD-modified PCL films; (C) co-cultured with SC-like ASC on untreated PCL films; (D) co-cultured with SC-like ASC on RGD-modified PCL films. The yellow arrows indicate the contact points between SC-like ASC and DRG, confirming their direct interaction in the co-culture system. Cells were maintained in culture for 3 days and fixed in 2.5 % glutaraldehyde. Following dehydration in graded ethanol series (50 %, 70 %, 90 %, 100 %), cells were finally rinsed in hexamethyldisilazane and dried before mounting on stubs and gold sputtering for SEM analysis. Images were acquired using a SEM (Zeiss EVO60, UK) with an accelerating voltage of 10kV. (Re-print with permission from de Luca et al.30. <a href="https://www.jove.com/files/ftp_upload/52543/52543fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration at JoVE.com

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) are structures containing the sensory neurons of the peripheral nervous system. When dissociated, they can be co-cultured with SC-like adipose-derived stem cells (ASC), providing a valuable model to study in vitro nerve regeneration and myelination, mimicking the in vivo environment at the injury site.

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the ...

dorsal-root-ganglia-neurons-differentiated-adipose-derived-stem-cells

Research

JoVE Journal

Neuroscience

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after injury (Teng et al., 2006). It is recognized that transcriptional programs essential for neurite and axonal outgrowth are 
reactivated upon injury in the PNS (Makwana et al., 2005). However the tools available to analyze neuronal gene regulation in vivo are limited and 
often challenging.
The dorsal root ganglia (DRG) offer an excellent injury model system because both the CNS and PNS are innervated by a 
bifurcated axon originating from the same soma. The ganglia represent a discrete collection of cell bodies where all transcriptional events occur, 
and thus provide a clearly defined region of transcriptional activity that can be easily and reproducibly removed from the animal. Injury of nerve 
fibers in the PNS (e.g. sciatic nerve), where axonal regeneration does occur, should reveal a set of transcriptional programs that are distinct from 
those responding to a similar injury in the CNS, where regeneration does not take place (e.g. spinal cord). Sites for transcription factor binding, 
histone and DNA modification resulting from injury to either PNS or CNS can be characterized using chromatin immunoprecipitation (ChIP). 
Here, we describe a ChIP protocol using fixed mouse DRG tissue following axonal injury. This powerful combination provides a means for characterizing the pro-regeneration chromatin environment necessary for promoting axonal regeneration.

We present a method for chromatin immunoprecipitation from dorsal root ganglia tissue following axonal injury. The approach can be used to identify specific transcription factor binding sites and epigenetic modification of histone and DNA important for the regeneration of injured axons in both the peripheral and central nervous system.

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after ...

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 ...

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory system is characterized by its ability to give rise to new neurons even in adult animals. This particular ability is partly due to the presence of OECs which create a favorable microenvironment for neurogenesis. This property of OECs has been used for cellular transplantation such as in spinal cord injury models. Although the peripheral nervous system has a greater capacity to regenerate after nerve injury than the central nervous system, complete sections induce misrouting during axonal regrowth in particular after facial of laryngeal nerve transection. Specifically, full sectioning of the recurrent laryngeal nerve (RLN) induces aberrant axonal regrowth resulting in synkinesis of the vocal cords. In this specific model, we showed that OECs transplantation efficiently increases axonal regrowth.
OECs are constituted of several subpopulations present in both the olfactory mucosa (OM-OECs) and the olfactory bulbs (OB-OECs). We present here a model of cellular transplantation based on the use of these different subpopulations of OECs in a RLN injury model. Using this paradigm, primary cultures of OB-OECs and OM-OECs were transplanted in Matrigel after section and anastomosis of the RLN. Two months after surgery, we evaluated transplanted animals by complementary analyses based on videolaryngoscopy, electromyography (EMG), and histological studies. First, videolaryngoscopy allowed us to evaluate laryngeal functions, in particular muscular cocontractions phenomena. Then, EMG analyses demonstrated richness and synchronization of muscular activities. Finally, histological studies based on toluidine blue staining allowed the quantification of the number and profile of myelinated fibers.
All together, we describe here how to isolate, culture, identify and transplant OECs from OM and OB after RLN section-anastomosis and how to evaluate and analyze the efficiency of these transplanted cells on axonal regrowth and laryngeal functions.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth of the primary olfactory neurons. This specific property can be used for cellular transplantation. We present here a model of cellular transplantation based on the use of OECs in a laryngeal nerve injury model.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory ...

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These neurotropic cancer cells have a unique ability to migrate unidirectionally along nerves towards the central nervous system (CNS). The dorsal root ganglia (DRG)/cancer cell model is a three dimensional (3D) in vitro model frequently used for studying the interaction between neural stroma and cancer cells. In this model, mouse or human cancer cell lines are grown in ECM adjacent to preparations of freshly dissociated cultured DRG. In this article, the DRG isolation protocol from mice, and implantation in petri dishes for co-culturing with pancreatic cancer cells are demonstrated. Five days after implantation, the cancer cells made contact with the DRG neurites. Later, these cells formed bridgeheads to facilitate more extensive polarized, neurotropic migration of cancer cells.

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

This video article shows the use of the dorsal root ganglia (DRG)/cancer cell model in pancreatic ductal adenocarcinoma.

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These ...

An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to bridge the lesion site, properly organized axonal alignment is required. Since demyelination after nerve injury strongly impairs the conductive capacity of surviving axons, remyelination is critical for successful functioning of regenerated nerves. Previously, we demonstrated that mesenchymal stem cells (MSCs) aligned on a pre-stretch induced anisotropic surface because the cells can sense a larger effective stiffness in the stretched direction than in the perpendicular direction. We also showed that an anisotropic surface arising from a mechanical pre-stretched surface similarly affects alignment, as well as growth and myelination of axons. Here, we provide a detailed protocol for preparing a pre-stretched anisotropic surface, the isolation and culture of dorsal root ganglion (DRG) neurons on a pre-stretched surface, and show the myelination behavior of a co-culture of DRG neurons with Schwann cells (SCs) on a pre-stretched surface.

This protocol describes the isolation of dorsal root ganglion (DRG) neurons isolated from rats and the culture of DRG neurons on a static pre-stretched cell culture system to enhance axon alignment, with subsequent co-culture of Schwann Cells (SCs) to promote myelination.

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to ...

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 ...

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral tissues, such as skin, muscle and visceral organs, as well as the spinal dorsal horn of the central nervous system. Sensory neurons transmit somatic sensation, including touch, pain, thermal, and proprioceptive sensations. Therefore, DRG primary cultures are widely used to study the cellular mechanisms of nociception, physiological functions of sensory neurons, and neural development. The cultured neurons can be applied in studies involving electrophysiology, signal transduction, neurotransmitter release, or calcium imaging. With DRG primary cultures, scientists may culture dissociated DRG neurons to monitor biochemical changes in single or multiple cells, overcoming many of the limitations associated with in vivo experiments. Compared to commercially available DRG-hybridoma cell lines or immortalized DRG neuronal cell lines, the composition and properties of the primary cells are much more similar to sensory neurons in tissue. However, due to the limited number of cultured DRG primary cells that can be isolated from a single animal, it is difficult to perform high-throughput screens for drug targeting studies. In the current article, procedures for DRG collection and culture are described. In addition, we demonstrate the treatment of cultured DRG cells with an agonist of neuropeptide FF receptor type 2 (NPFFR2) to induce the release of peptide neurotransmitters (calcitonin gene-related peptide (CRGP) and substance P (SP)).

Dorsal root ganglia (DRG) primary cultures are frequently used to study physiological functions or pathology-related events in sensory neurons. Here, we demonstrate the use of lumbar DRG cultures to detect the release of neurotransmitters after neuropeptide FF receptor type 2 stimulation with a selective agonist.

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral ...

Facial Nerve Surgery in the Rat Model to Study Axonal Inhibition and Regeneration

This protocol describes consistent and reproducible methods to study axonal regeneration and inhibition in a rat facial nerve injury model. The facial nerve can be manipulated along its entire length, from its intracranial segment to its extratemporal course. There are three primary types of nerve injury used for the experimental study of regenerative properties: nerve crush, transection, and nerve gap. The range of possible interventions is vast, including surgical manipulation of the nerve, delivery of neuroactive reagents or cells, and either central or end-organ manipulations. Advantages of this model for studying nerve regeneration include simplicity, reproducibility, interspecies consistency, reliable survival rates of the rat, and an increased anatomic size relative to murine models. Its limitations involve a more limited genetic manipulation versus the mouse model and the superlative regenerative capability of the rat, such that the facial nerve scientist must carefully assess time points for recovery and whether to translate results to higher animals and human studies. The rat model for facial nerve injury allows for functional, electrophysiological, and histomorphometric parameters for the interpretation and comparison of nerve regeneration. It thereby boasts tremendous potential toward furthering the understanding and treatment of the devastating consequences of facial nerve injury in human patients.

This protocol describes a reproducible approach to facial nerve surgery in the rat model, including descriptions of various inducible patterns of injury.

This protocol describes consistent and reproducible methods to study axonal regeneration and inhibition in a rat facial nerve injury model. The facial ...

A Scalable Model to Study the Effects of Blunt-Force Injury in Adult Zebrafish

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and heterogenous secondary effects. While there is no mechanism to replace or regenerate the lost neurons following a TBI in humans, zebrafish possess the ability to regenerate neurons throughout their body, including the brain. To examine the breadth of pathologies exhibited in zebrafish following a blunt-force TBI and to study the mechanisms underlying the subsequent neuronal regenerative response, we modified the commonly used rodent Marmarou weight drop for the use in adult zebrafish. Our simple blunt-force TBI model is scalable, inducing a mild, moderate, or severe TBI, and recapitulates many of the phenotypes observed following human TBI, such as contact- and post-traumatic seizures, edema, subdural and intracerebral hematomas, and cognitive impairments, each displayed in an injury severity-dependent manner. TBI sequelae, which begin to appear within minutes of the injury, subside and return to near undamaged control levels within 7 days post-injury. The regenerative process begins as early as 48 hours post-injury (hpi), with the peak cell proliferation observed by 60 hpi. Thus, our zebrafish blunt-force TBI model produces characteristic primary and secondary injury TBI pathologies similar to human TBI, which allows for investigating disease onset and progression, along with the mechanisms of neuronal regeneration that is unique to zebrafish.

We modified the Marmarou weight drop model for adult zebrafish to examine a breadth of pathologies following blunt-force traumatic brain injury (TBI) and the mechanisms underlying subsequent neuronal regeneration. This blunt-force TBI model is scalable, induces a mild, moderate, or severe TBI, and recapitulates injury heterogeneity observed in human TBI.