Name: dorsal root ganglia isolation and primary culture to study neurotransmitter release
Uploaded: 2018-10-06T13:00:00
Description: Watch this Scientific Journal Video about Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release at JoVE.com

We thank Dr. M. Calkins for English editing. This work was supported by the Chang Gung Memorial Hospital (CMRPD1F0482), Chang Gung University, Healthy Aging Research Center (EMRPD1G0171) and Ministry of Science and Technology (105-2320-B-182-012-MY2).

All methods described herein that use experimental animals were approved by the Institutional Animal Care and Use Committee (IACUC) of Chang Gung University (CGU 13-014).
1. Collect Lumbar DRG from Experimental Rats
<ol>
	<li>Use 2 to 3 week-old Sprague-Dawley (SD) rats for lumbar DRG collection. 
	NOTE: DRG neurons collected from rats over 4 weeks of age do not grow well under the culture conditions described herein.</li>
	<li>Sterilize all surgical instruments in an autoclave.</li>
	<...

<ol>
	<li>Bear, M. F., Connors, B. W., Paradiso, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neuroscience: exploring the brain. , (2007).</li><li>Hunt, S. P., Mantyh, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+dynamics+of+pain+control.">The molecular dynamics of pain control.</a> Nat Rev Neurosci. 2 (2), 83-91 (2001).</li><li>Kandel, E. R., Schwartz, J. H., Jessell, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles of neural science. , (2000).</li><li>Julius, D., Basbaum, A. 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S., Santiago, M. F., Marques, S. A., Allodi, S., Martinez, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+among+satellite+glial+cells+in+dorsal+root+ganglia+of+the+rat.">Diversity among satellite glial cells in dorsal root ganglia of the rat.</a> Braz J Med Biol Res. 41 (11), 1011-1017 (2008).</li><li>Costa, F. A., Moreira Neto, F. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Satellite+glial+cells+in+sensory+ganglia:+its+role+in+pain.">Satellite glial cells in sensory ganglia: its role in pain.</a> Rev Bras Anestesiol. 65 (1), 73-81 (2015).</li><li>Malin, S. A., Davis, B. M., Molliver, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+dissociated+sensory+neuron+cultures+and+considerations+for+their+use+in+studying+neuronal+function+and+plasticity.">Production of dissociated sensory neuron cultures and considerations for their use in studying neuronal function and plasticity.</a> Nat Protoc. 2 (1), 152-160 (2007).</li><li>Lin, Y. T., Ro, L. S., Wang, H. L., Chen, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Up-regulation+of+dorsal+root+ganglia+BDNF+and+trkB+receptor+in+inflammatory+pain:+an+in+vivo+and+in+vitro+study.">Up-regulation of dorsal root ganglia BDNF and trkB receptor in inflammatory pain: an in vivo and in vitro study.</a> J Neuroinflammation. 8, 126 (2011).</li><li>Liem, L., van Dongen, E., Huygen, F. J., Staats, P., Kramer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Dorsal+Root+Ganglion+as+a+Therapeutic+Target+for+Chronic+Pain.">The Dorsal Root Ganglion as a Therapeutic Target for Chronic Pain.</a> Reg Anesth Pain Med. 41 (4), 511-519 (2016).</li><li>Lee, J. S., Han, J. S., Lee, K., Bang, J., Lee, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+peripheral+and+central+mechanisms+underlying+itch.">The peripheral and central mechanisms underlying itch.</a> BMB Rep. 49 (9), 474-487 (2016).</li><li>Valtcheva, M. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+extraction+of+human+dorsal+root+ganglia+from+organ+donors+and+preparation+of+primary+sensory+neuron+cultures.">Surgical extraction of human dorsal root ganglia from organ donors and preparation of primary sensory neuron cultures.</a> Nat Protoc. 11 (10), 1877-1888 (2016).</li><li>Melli, G., Hoke, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsal+Root+Ganglia+Sensory+Neuronal+Cultures:+a+tool+for+drug+discovery+for+peripheral+neuropathies.">Dorsal Root Ganglia Sensory Neuronal Cultures: a tool for drug discovery for peripheral neuropathies.</a> Expert Opin Drug Discov. 4 (10), 1035-1045 (2009).</li><li>Yin, K., Baillie, G. J., Vetter, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+cell+lines+as+model+dorsal+root+ganglion+neurons:+A+transcriptomic+comparison.">Neuronal cell lines as model dorsal root ganglion neurons: A transcriptomic comparison.</a> Mol Pain. 12, (2016).</li><li>Chen, W., Mi, R., Haughey, N., Oz, M., Hoke, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalization+and+characterization+of+a+nociceptive+dorsal+root+ganglion+sensory+neuronal+line.">Immortalization and characterization of a nociceptive dorsal root ganglion sensory neuronal line.</a> J Peripher Nerv Syst. 12 (2), 121-130 (2007).</li><li>Doran, C., Chetrit, J., Holley, M. C., Grundy, D., Nassar, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+DRG+Cell+Line+with+Properties+of+Nociceptors.">Mouse DRG Cell Line with Properties of Nociceptors.</a> PLoS One. 10 (6), e0128670 (2015).</li><li>Gouarderes, C., Roumy, M., Advokat, C., Jhamandas, K., Zajac, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+localization+of+neuropeptide+FF+receptors+in+the+rat+dorsal+horn.">Dual localization of neuropeptide FF receptors in the rat dorsal horn.</a> Synapse. 35 (1), 45-52 (2000).</li><li>Lin, Y. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+NPFFR2+leads+to+hyperalgesia+through+the+spinal+inflammatory+mediator+CGRP+in+mice.">Activation of NPFFR2 leads to hyperalgesia through the spinal inflammatory mediator CGRP in mice.</a> Exp Neurol. 291, 62-73 (2017).</li><li>Yang, H. Y., Tao, T., Iadarola, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulatory+role+of+neuropeptide+FF+system+in+nociception+and+opiate+analgesia.">Modulatory role of neuropeptide FF system in nociception and opiate analgesia.</a> Neuropeptides. 42 (1), 1-18 (2008).</li><li>Takeda, M., Takahashi, M., Matsumoto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+the+activation+of+satellite+glia+in+sensory+ganglia+to+pathological+pain.">Contribution of the activation of satellite glia in sensory ganglia to pathological pain.</a> Neurosci Biobehav Rev. 33 (6), 784-792 (2009).</li></ol>

In the present article, we demonstrate the collection, enzyme-dissociation, and culture of rat lumbar DRG. With the neurotrophic support from NGF, the axons of DRG neurons extended within 3 days after cell seeding. The extended axons were clearly observable after cells were stained for CGRP protein, which is synthesized in the cell soma and transported along the axon fibers. The processes of satellite cells also extended, allowing these dividing glial cells to surround the neurons within days. The primary DRG cells grown...

The cell bodies of sensory neurons are contained within DRG. These neurons are pseudo-unipolar and innervate both peripheral tissues and the central nervous system. The peripheral nerve endings of sensory neurons are found in muscle, skin, visceral organs, and bone, among other tissues. They transmit peripheral sensation signals to nerve endings in the spinal dorsal horn and the signals are then transmitted to the brain via different ascending pathways of somatic sensation1,2. Somatic sensation enables the body to feel (i.e., touch, pain, and thermal sensations) and perceive movement and spatial orientation (proprioceptive sensations)1,3. There are four subclasses of primary afferent axons, including group I (A&#945;) fibers that respond to proprioception of skeletal muscles, group II (A&#946;) fibers that respond to mechanoreceptors of the skin, and group III (A&#948;) and group V (C) fibers that respond to pain and temperature. Only the C fibers are unmyelinated, while the rest are myelinated to different degrees.
Nociceptors are primary sensory neurons, which are activated by noxious stimuli (mechanical, thermal, and chemical stimulation) that carry potential for tissue damage. These neurons are composed of myelinated A&#948; fibers and unmyelinated C fibers1,4. The A&#948; fibers express the receptors for nerve growth factor (NGF, trkA receptor), CGRP, and SP. The C fibers are classified as either peptidergic and non-peptidergic C fibers. On the other hand, the non-peptidergic C fibers express the receptors for glial-derived neurotrophic factor (GDNF, RET, and GFR receptors), isolectin IB4, and ATP-gated ion channel subtype (P2X3)5,6,7. Nociceptors can be distinguished by the expression of ion channels and activated by neurotrophic factors, cytokines, neuropeptides, ATP, or other chemical compounds8. Upon stimulation, neurotransmitters, including CGRP, SP, and glutamate may be released from sensory neuron terminals in the spinal dorsal horn to transmit nociceptive signals2. DRG are not only composed of neurons, but also contain satellite glial cells. Satellite cells surround the sensory neurons and provide mechanical and metabolic support9,10. Interestingly, there is a growing body of evidence indicating that satellite glial cells in the DRG may be involved in regulating pain sensation11.
Sensory neurons have been reported to be the most frequently used primary neuronal cells12 and have been utilized for electrophysiology, signal transduction, and neurotransmitter release studies. They are also commonly used to explore the cellular mechanisms of neuronal development, inflammatory pain, neuropathic pain, skin sensation (like itch), and axon outgrowth12,13,14,15. DRG primary cultures can be cultured as dissociated neurons to assess biochemical changes in single or multiple cells, allowing scientists to perform studies that cannot be performed in experimental subjects. Recently, DRG were successfully cultured from human organ donors which might greatly benefit translational research16. On the other hand, sensory neurons can also be cultured as DRG explants. The DRG explants preserve the original tissue architecture of the neurons, including Schwann cells and satellite glial cells, and are especially useful to study interactions between neuronal and non-neuronal cells17. DRG primary cultures can be easily prepared within 2.5 h. The cell composition and properties are highly reflective of the source DRG, and as such, specific DRG (lumbar or thoracic DRG) can be collected according to experimental demands. Cultures of embryonic and neonatal DRG neurons require NGF to survive and induce axon outgrowth, but cultures of adult neurons do not require the addition of neurotrophic factors to the media12,17. There are also commercially available DRG-hybridoma cell lines such as ND7/23 and F11, which do not require the use of experimental animals. However, the lack of the transient receptor potential cation channel subfamily V member 1 (TRPV1) expression (an important marker for small sensory nociceptive neurons) and incongruent gene expression profiles limit their applications18. Recently, immortalized DRG neuronal cell lines have been derived from rat (50B11)19 and mouse (MED17.11)20, which are suitable for use in high-throughput screens for drug targeting studies. However, gene expression profiling for these cell lines has yet to be performed. Thus, the validation experiments comparing these immortalized cells to sensory neurons are still ongoing.
NPFFR2 is synthesized in the DRG and translocated to the sensory nerve terminals in the spinal dorsal horn21. In this article, we provide a protocol for culturing lumbar DRG cells and treating them with an agonist of NPFFR2 to induce the release of neurotransmitters, CGRP and SP. The dependence on NPFFR2 is further tested using NPFFR2 small interfering RNA (siRNA), which may be transfected into the cultured DRG cells.

<table>
	<tbody>
		<tr>
			<td>Mixture of tiletamine and zolazepam (Zoletil)</td>
			<td>Virbac</td>
			<td>Zoletil 50</td>
			<td>anaesthetic</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Biological Industries</td>
			<td>04-001-1</td>
			<td>Culture Medium</td>
		</tr>
		<tr>
			<td>sodium pyruvate</td>
			<td>Sigma</td>
			<td>S8636</td>
			<td>Culture Medium</td>
		</tr>
		<tr>
			<td>penicillin/streptomycin</td>
			<td>Biological Industries</td>
			<td>03-033-1</td>
			<td>Culture Medium</td>
		</tr>
		<tr>
			<td>DMEM-F12</td>
			<td>Invitrogen</td>
			<td>12400024</td>
			<td>Culture Medium</td>
		</tr>
		<tr>
			<td>Poly-l-lysine</td>
			<td>Sigma</td>
			<td>P9011</td>
			<td>Coating dish</td>
		</tr>
		<tr>
			<td>Collagenase IA</td>
			<td>Sigma</td>
			<td>9001-12-1</td>
			<td>Enzyme digestion</td>
		</tr>
		<tr>
			<td>Hank&#39;s balanced salt solution</td>
			<td>Invitrogen</td>
			<td>14170-112</td>
			<td>Culture Medium</td>
		</tr>
		<tr>
			<td>Trypsin EDTA</td>
			<td>Biological Industries</td>
			<td>03-051-5</td>
			<td>Enzyme digestion</td>
		</tr>
		<tr>
			<td>Pasteur pipette</td>
			<td>Hilgenberg</td>
			<td>3150102</td>
			<td>Cell trituration</td>
		</tr>
		<tr>
			<td>Cytarabine (Ara-C)</td>
			<td>Sigma</td>
			<td>C6645</td>
			<td>Culture Medium</td>
		</tr>
		<tr>
			<td>NGF</td>
			<td>Millipore</td>
			<td>NC011</td>
			<td>Culture Medium</td>
		</tr>
		<tr>
			<td>NPFFR2 siRNA</td>
			<td>Dharmacon</td>
			<td>L-099691-02-0005</td>
			<td>Transfection</td>
		</tr>
		<tr>
			<td>Non-targeting siRNA</td>
			<td>Dharmacon</td>
			<td>L-001810-10-05</td>
			<td>Transfection</td>
		</tr>
		<tr>
			<td>NeuroPORTER Reagent</td>
			<td>Genlantis</td>
			<td>T400150</td>
			<td>Transfection reagent</td>
		</tr>
		<tr>
			<td>dNPA</td>
			<td>Genemed Synthesis</td>
			<td>N/A</td>
			<td>NPFFR2 agonist</td>
		</tr>
		<tr>
			<td>CGRP ELISA</td>
			<td>Cayman</td>
			<td>589001</td>
			<td>EIA</td>
		</tr>
		<tr>
			<td>SP ELISA</td>
			<td>Cayman</td>
			<td>583751</td>
			<td>EIA</td>
		</tr>
		<tr>
			<td>CGRP antibody</td>
			<td>Calbiochem</td>
			<td>PC205L</td>
			<td>IHC</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Roche</td>
			<td>10236276001</td>
			<td>IHC</td>
		</tr>
	</tbody>
</table>

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)).

Rat lumbar DRG neurons, cultured in a 24-well plate, were grown in culture medium with additional Ara-C to inhibit glial cell proliferation and NGF to support neuronal growth. The morphology of living DRG cells was observed. As shown in Figure 3, the cell body of a single neuron was attached on the bottom of a dish at Day 1 and selected for observation. Axon growth was monitored from Day 1&#8211;3. The glial cells duplicated and extended processes to surround...

Watch this Scientific Journal Video about Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release at JoVE.com

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

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 ...

This method can help to answer key questions in the sensory research field, such as peripheral nociception. The main advantage of this technical is that investigating the cellular mechanism of sensory neurons with the model that most simulates to physiological condition. Collect the lumbar dorsal root ganglia from the trunk of a two to three week old rat.Begin by making two cuts along the sides of the spinal column and one lateral cut to mark the rostral extent of the lumbar spine. Then, use bone-cutting forceps to remove the dorsal muscles of the spine. Next, remove the dorsal portion of the vertebrae and expose the spinal cord.Then use dissection scissors and forceps to remove the spinal cord. Identify the lumbar DRG by counting vertebrae from the last rib. This diagram shows the vertebrae positions.Use micro scissors to collect the 12 bilateral lumbar DRG from L1 to L6.Remove any attached neuronal fibers to improve the purity of the culture. Transfer each lumbar DRG to a 35 millimeter culture dish containing two milliliters of ice cold serum-free medium. Transfer the DRG-containing 35 millimeter dish into a laminar hood, and wash the DRG with serum-free medium three times by pipette.Use sterile tweezers to move the DRGs to a new 35 millimeter culture dish containing two milliliters of sterile collagenase type 1A. Place the tissue in collagenase solution in the 37 degree Celsius tissue culture incubator for 30 minutes to begin dissociation of the cells. Following the incubation, remove the collagenase solution and wash the DRG three times in two milliliters of Hank's balanced salt solution.Next, add two milliliters of prewarmed 0.05%trypsin EDTA to the dish, and digest the DRG in the incubator for 30 minutes as before. After digestion, use a glass pipette to transfer the two milliliters of DRG-containing solution to a 15 milliliter centrifuge tube. The DRG might stick to the glass pipette so this step should be performed with care.The actual loss can be avoid by keeping the DRG-containing solution in the taper end of the glass pipette, and transferring the solution into the centrifuge tube slowly but without pause. Next, centrifuge the solution at 290 times G for five minutes at four degrees Celsius. After centrifuging, remove the supernatant and add another two milliliters of serum-free medium to resuspend the DRG.Repeat the wash twice, using two milliliters of prewarmed culture medium to resuspend the tissue after the final centrifugation. Use the previously prepared sterile flame polished pipette to manually triturate the DRG approximately 60 times. Next, remove a poly-L-lysine coated 24 well plate containing one milliliter of culture medium per well from the CO2 incubator.Aspirate the incubated culture medium from the dish. Seed the DRG cells from one rat into four of the wells. This gives a seeding density of approximately 500, 000 cells per well.The following day, replace the culture medium with medium supplemented with 10 micromolar AraC, and 100 nanograms per milliliter NGF. On day three after cell plating, change the medium to 0.5 milliliters of prewarmed serum-free medium, and incubate for one hour. During the incubation, add 50 millimolar siRNA in one microliter of RNase-free water to 12.5 microliters of serum-free medium per transfection.Then pipette 2.5 microliters of transfection reagent into 10 microliters of serum-free medium for each transfection. Mix both solutions by pipette, and incubate this mixed transfection solution for 10 minutes at room temperature. After 10 minutes, add the transfection solution into the wells of the DRG-containing 24 well plate and mix by shaking gently.Following a six hour incubation, add 0.5 milliliters of culture medium with 20%FBS, 10 micromolar AraC, and 100 nanograms per milliliter NGF into each well of cells. Finally, incubate the DRG in a 37 degree Celsius CO2 incubator for another 66 hours. On day six after plating, and 72 hours after siRNA transfection, change the culture medium to 200 microliters of serum-free medium.Following a 30 minute incubation, add one microliter of the stimulation chemical and gently mix by pipetting. The NPFFR2 agonist, dNPA, and corresponding vehicle are used here. After incubating for the required period, collect the culture medium from the culture dish, and centrifuge at 5000 times G for five minutes at four degrees Celsius to remove any suspended impurities.Collect the supernatant from the centrifugation and dilute with phosphate-buffered saline as needed. Assay the levels of neurotransmitters with commercially available enzyme immunoassay kits. On day one, the cell body of a single neuron indicated by the arrow was attached on the bottom of a dish.A glial cell indicated by an arrowhead is also present. The glial cells duplicated and extended processes to surround the cell body of the sensory neuron on day two, a process that was even more prominent on day three. In another culture, CGRP protein was stained to reveal the shape of neurons.CGRP protein staining appears in the cytoplasm and axons of sensory neurons. The nuclear morphologies of neurons and glial cells are distinct when stained with DAPI. The neurons have a larger and more rounded nucleus than glial cells.By comparison, the nuclei of glial are more oval in shape. Once mastered, this technique can be done in two and half hours if it is performed properly. After watching this video, you should have a good understanding of how to dissect and culture the dorsal root ganglion, and use it as a tool to investigate underlying physiological functions.Don't forget that working with primary cells can be much more difficult than working with regular cell lines, and being gentle is always the best way when performing these procedure.

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Research

JoVE Journal

Neuroscience

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 ...

Live Imaging of Dorsal Root Axons after Rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the interface between the CNS and PNS. Our understanding of the molecular and cellular events that prevent regeneration at DREZ is incomplete, in part because complex changes associated with nerve injury have been deduced from postmortem analyses. Dynamic cellular processes, such as axon regeneration, are best studied with techniques that capture real-time events with multiple observations of each living animal. Our ability to monitor neurons serially in vivo has increased dramatically owing to revolutionary innovations in optics and mouse transgenics. Several lines of thy1-GFP transgenic mice, in which subsets of neurons are genetically labeled in distinct fluorescent colors, permit individual neurons to be imaged in vivo1. These mice have been used extensively for in vivo imaging of muscle2-4 and brain5-7, and have provided novel insights into physiological mechanisms that static analyses could not have resolved. Imaging studies of neurons in living spinal cord have only recently begun. Lichtman and his colleagues first demonstrated their feasibility by tracking injured dorsal column (DC) axons with wide-field microscopy8,9. Multi-photon in vivo imaging of deeply positioned DC axons, microglia and blood vessels has also been accomplished10. Over the last few years, we have pioneered in applying in vivo imaging to monitor regeneration of DR axons using wide-field microscopy and H line of thy1-YFP mice. These studies have led us to a novel hypothesis about why DR axons are prevented from regenerating within the spinal cord11.
In H line of thy1-YFP mice, distinct YFP+ axons are superficially positioned, which allows several axons to be monitored simultaneously. We have learned that DR axons arriving at DREZ are better imaged in lumbar than in cervical spinal cord. In the present report we describe several strategies that we have found useful to assure successful long-term and repeated imaging of regenerating DR axons. These include methods that eliminate repeated intubation and respiratory interruption, minimize surgery-associated stress and scar formation, and acquire stable images at high resolution without phototoxicity.

An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS.

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

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 ...

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.

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

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 ...

Hydraulic Extrusion of the Spinal Cord and Isolation of Dorsal Root Ganglia in Rodents

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may result in damage to the spinal cord caused by the dissection process. Here, we show how the spinal cord can be extruded using hydraulic pressure. Handling time is significantly reduced to only a few minutes, likely decreasing protein damage. The low risk of damage to the spinal cord tissue improves subsequent immunohistochemical analysis. By performing hydraulic spinal cord extrusion instead of traditional laminectomy, the rodents can further be used for DRG isolation, thereby lowering the number of animals and allowing analysis across tissues from the same rodent. We demonstrate a consistent method to identify and isolate the DRGs according to their localization relative to the costae. It is, however, important to adjust this method to the particular animal used, as the number of spinal cord segments, both thoracic and lumbar, may vary according to animal type and strain. In addition, we illustrate further processing examples of the isolated tissues.

Here, we present a protocol for hydraulic extrusion of the spinal cord as well as identification and isolation of specific dorsal root ganglia (DRGs) in the same rodent. Compared to standard spinal cord isolation methods, this method is significantly faster and reduces the risk of tissue damage.

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may ...

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 ...

Laminotomy for Lumbar Dorsal Root Ganglion Access and Injection in Swine

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive targets for injection of novel therapeutics aimed at treating chronic pain. In small animal models, laminectomy has been used to facilitate DRG injection because it involves surgical removal of the vertebral bone surrounding each DRG. We demonstrate a technique for intraganglionic injection of lumbar DRG in a large animal species, namely, swine. Laminotomy is performed to allow direct access to DRG using standard neurosurgical techniques, instruments, and materials. Compared with more extensive bone removal via laminectomy, we implement laminotomy to conserve spinal anatomy while achieving sufficient DRG access. Intraoperative progress of DRG injection is monitored using a non-toxic dye. Following euthanasia on post-operative day 21, the success of injection is determined by histology for intraganglionic distribution of 4&#39;,6-diamidino-2-phenylindole (DAPI). We inject a biologically inactive solution to demonstrate the protocol. This method could be applied in future preclinical studies to target therapeutic solutions to DRG. Our methodology should facilitate testing the translatability of intraganglionic small animal paradigms in a large animal species. Additionally, this protocol may serve as a key resource for those planning preclinical studies of DRG injection in swine.

We describe a method for laminotomy in swine that provides access to lumbar dorsal root ganglia (DRG) for intraganglionic injection. Injection progress is monitored intraoperatively and histologically confirmed up to 21 days after surgery. This protocol could be used for future preclinical studies involving DRG injection.

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive ...

Rapid Isolation of Dorsal Root Ganglion Macrophages

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after peripheral nerve injury. Peripheral monocytic cells, including macrophages, are known to respond to a tissue injury through phagocytosis, antigen presentation, and cytokine release. Emerging evidence has implicated the contribution of dorsal root ganglia macrophages to neuropathic pain development and axonal repair in the context of nerve injury. Rapidly phenotyping (or &#8220;rapid isolation of&#8221;) the response of dorsal root ganglia macrophages in the context of nerve injury is desired to identify the unknown neuroimmune factors. Here we demonstrate how our lab rapidly and effectively isolates macrophages from the dorsal root ganglia using an enzyme-free mechanical dissociation protocol. The samples are kept on ice throughout to limit cellular stress. This protocol is far less time consuming compared to the standard enzymatic protocol and has been routinely used for our Fluorescence-activated Cell Sorting analysis.

Here we present a mechanical dissociation protocol to rapidly isolate macrophages from the dorsal root ganglion for phenotyping and functional analysis.

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after ...

Enrichment of Adult Mouse Dorsal Root Ganglia Neuron Cultures by Immunopanning

Dorsal root ganglia (DRGs) are peripheral structures adjacent to the dorsal horn of the spinal cord, which house the cell bodies of sensory neurons as well as various other cell types. Published culture protocols often refer to whole dissociated DRG cultures as being neuronal, despite the presence of fibroblasts, Schwann cells, macrophages, and lymphocytes. While these whole DRG cultures are sufficient for imaging applications where neurons can be discerned based on morphology or staining, protein or RNA homogenates collected from these cultures are not primarily neuronal in origin. Here, we describe an immunopanning sequence for cultured mouse DRGs. The goal of this method is to enrich DRG cultures for neurons by removing other cell types. Immunopanning refers to a method of removing cell types by adhering antibodies to cell culture dishes. Using these dishes, we can negatively select against and reduce the number of fibroblasts, immune cells, and Schwann cells in culture. This method allows us to increase the percentage of neurons in cultures.

This paper describes an immunopanning protocol for adult mouse dorsal root ganglia. By adhering antibodies to culture plates, we can negatively select and remove non-neuronal cells. We show that the cultures are enriched for neurons using this protocol, allowing for an in-depth study of neuronal responses to manipulation.

Dorsal root ganglia (DRGs) are peripheral structures adjacent to the dorsal horn of the spinal cord, which house the cell bodies of sensory neurons as ...