We sincerely thank the Imaging core-facility at Midwestern University (MWU) and the group of students [Chanmoly Seng, Christopher Dipollina, Darryl Giambalvo, and Casey Sigerson] for their contributions in cell culture and imaging work. This research work was supported by the MWU&#39;s Intramural grant funding to M.F. and research start-up funds to V.T.

All the procedures including the use of the animals have been approved by the institutional review board-approved protocols (IACUC- Midwestern University).
1. Harvesting DRG from Mouse Embryos
<ol>
	<li>Euthanize the adult mice by asphyxiation method (CO2) followed by decapitation. Immediately proceed to surgically remove the vertebral column.
	<ol>
		<li>Expose the vertebral column by cutting down the skin layer dorsally using fine scissors. Isolate the vertebral...

<ol>
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B., Boyer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+effects+of+nerve+injury+on+estimates+of+sensory+neuron+number+in+juvenile+bullfrog.">Transient effects of nerve injury on estimates of sensory neuron number in juvenile bullfrog.</a> J Comp Neurol. 410 (2), 171-177 (1999).</li><li>Geuna, S., Borrione, P., Poncino, A., Giacobini-Robecchi, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+and+morphometrical+changes+in+dorsal+root+ganglion+neurons+innervating+the+regenerated+lizard+tail.">Morphological and morphometrical changes in dorsal root ganglion neurons innervating the regenerated lizard tail.</a> Int J Dev Neurosci. 16 (2), 85-95 (1998).</li><li>La Forte, R. A., Melville, S., Chung, K., Coggeshall, R. 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S., 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=Functional+and+morphological+assessment+of+a+standardized+rat+sciatic+nerve+crush+injury+with+a+non-serrated+clamp.">Functional and morphological assessment of a standardized rat sciatic nerve crush injury with a non-serrated clamp.</a> J Neurotrauma. 21 (11), 1652-1670 (2004).</li><li>Hanack, C., 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=GABA+blocks+pathological+but+not+acute+TRPV1+pain+signals.">GABA blocks pathological but not acute TRPV1 pain signals.</a> Cell. 160 (4), 759-770 (2015).</li><li>Pagadala, P., 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=Loss+of+NR1+subunit+of+NMDARs+in+primary+sensory+neurons+leads+to+hyperexcitability+and+pain+hypersensitivity:+involvement+of+Ca(2+)-activated+small+conductance+potassium+channels.">Loss of NR1 subunit of NMDARs in primary sensory neurons leads to hyperexcitability and pain hypersensitivity: involvement of Ca(2+)-activated small conductance potassium channels.</a> J Neurosci. 33 (33), 13425-13430 (2013).</li><li>Zhang, X. L., Albers, K. M., Gold, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation-induced+increase+in+nicotinic+acetylcholine+receptor+current+in+cutaneous+nociceptive+DRG+neurons+from+the+adult+rat.">Inflammation-induced increase in nicotinic acetylcholine receptor current in cutaneous nociceptive DRG neurons from the adult rat.</a> Neuroscience. 284, 483-499 (2015).</li><li>Zhu, Y., Lu, S. G., Gold, M. 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The authors have nothing to disclosure.

The ex vivo DRG model is extremely useful to investigate a wide spectrum of events such as neuron-glia interaction as well as the effect of the microenvironment on both neuronal and glial metabolism37. Further, the DRG-model could be used as a cost-effective tool to address relevant questions regarding pathogenic mechanism and associated markers by developing ex vivo systems for acute chronic and latent phase of infection or in a given disease. In addition, a screening library of...

In this manuscript, we report a method to obtain an organotypic ex vivo model of a mouse derived DRG model system as a preserved tissue-like microenvironment to investigate a wide range of biological responses to PNS insults ranging from neuron-glial interaction, neuroplasticity, inflammatory markers, to viral infection. In addition, we further developed a protocol to create a primary co-culture of DRG-derived single sensory neurons and satellite cells.
The DRG are satellite gray-matter-units located outside the central nervous system (CNS) along the dorsal spinal roots of spinal nerves. The DRG, located in proximity of intervertebral foramina, house pseudounipolar sensory neurons and satellite glial cells. The pseudounipolar neurons feature a single neurite that splits into a peripheral process carrying somatic and visceral inputs from peripheral targets to the cell body, and a central process that submits sensory information from the cell body into the CNS. A connective capsule defines and isolates this peripheral cluster of neurons and glial cells from the CNS. No postnatal cell migration to or from the DRG has ever been described and a local stem cell niche is responsible for neurogenic events occurring throughout life1. Therefore, this model is particularly suitable to study adult neurogenesis, axonogenesis, response to traumatic lesion, and cell death2,3,4,5,6,7,8,9 .
In the field of neuroregeneration, the DRG harvested from in vivo and explanted in vitro reproduces axonotmesis, an injury condition in which axons are fully severed and the neuronal cell body is disconnected from the innervated target10,11. It is well known that peripheral nerve injury can cause decreased and increased gene expression in the DRG and many of these changes are a result of regenerative processes but many may also be a result of immune response or another response from non-neuronal cells. By using an ex vivo system of isolated DRG, some of this complexity is removed and mechanistic pathways can be more easily investigated.
Besides its central role in conveying sensory inputs to the CNS, the abundance of receptors for many neurotransmitters including GABA12,13,14,15 at the level of the neuronal soma as well as evidence of interneuronal cross-excitation may suggest that DRG are sophisticated preliminary integrators of sensory inputs16,17. These new findings confer to the DRG explant the characteristics of a mini-neuronal network system similar to other &#34;mini-brain&#34; models, which are nervous-tissue-specific organoids used for broader experimental fields of investigation and therapeutic approach to neurological diseases18,19. These evidences together with the fact that the DRG is a discrete and well-defined cluster of neuronal tissue surrounded by a connective capsule, make it a suitable organ for ex vivo transplantation.
Culturing mouse DRG presents an attractive multicellular option to model human pathophysiologies due to structural and genetic similarities between the species. Additionally, a large repository of transgenic mouse strains is highly conducive to future mechanistic studies. Neurite extension both during development and after injury requires mechanical interactions between growth cone and substrate20,21. Nano- and micro-patterned substrates have been used as tools to direct neurite outgrowth and demonstrate their capacity to respond to topographical features in their microenvironments. Neurons have been shown to survive, adhere, migrate, and orient their axons to navigate surface features such as grooves in substrates22,23. However, these studies have typically utilized cultured cell lines and it is difficult to predict how primary neuronal cells will respond to well-defined, physical cues in vivo or ex vivo.
The ex vivo explant model of mouse DRG used for this proposal mimics the real cell-cell interaction and biochemical cues surrounding growing axons. Among many different experimental paradigms ranging from axonal regeneration, neurosphere production, to neuroinflammation, the DRG explant model continues to serve as a valuable tool to investigate the viral infection and latency aspect within sensory ganglia24,25,26,27.
The nervous system (NS) in general is target for viral infections28,29,30. Most viruses infect epithelial and endothelial cell surfaces and make their way from the surface tissue to the NS via peripheral nerve sensory and motor fibers. In particular, the herpes simplex virus type 1 (HSV-1) after an initial infection in epithelial cells establishes a life-long latency in the sensory ganglia preferably, the DRG of the PNS31,32. HSV-1 neuroptropic capability of infecting the PNS ultimately leads to neurological diseases33.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adult Mice NIH/Swiss</td>
			<td>Harlan Laboratories</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>35mm petri dish</td>
			<td>Cell Treat</td>
			<td>229635</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel ECM</td>
			<td>Sigma-Aldrich</td>
			<td>E1270</td>
			<td>gelatinous protein mixture</td>
		</tr>
		<tr>
			<td>F12 Media</td>
			<td>Gibco</td>
			<td>11765-054</td>
			<td>*Part of SFM media</td>
		</tr>
		<tr>
			<td>Collagenase IV</td>
			<td>Sigma-Aldrich</td>
			<td>C5138</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma-Aldrich</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Sigma-Aldrich</td>
			<td>F6178</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22um filter</td>
			<td>BD Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal media</td>
			<td>Gibco</td>
			<td>10888-022</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td>Supplement for neuronal culture</td>
		</tr>
		<tr>
			<td>PSN antibiotics</td>
			<td>Gibco</td>
			<td>15640-055</td>
			<td>*Part of SFM media 
			Antibiotic mixture</td>
		</tr>
		<tr>
			<td>L-glutamate</td>
			<td>Sigma-Aldrich</td>
			<td>G7513</td>
			<td>*Part of SFM media</td>
		</tr>
		<tr>
			<td>NGF</td>
			<td>Alomone Labs</td>
			<td>N-100</td>
			<td>Nerve growth factor</td>
		</tr>
		<tr>
			<td>Laminin coated coverslide</td>
			<td>Neuvitro</td>
			<td>GG-14-Laminin</td>
			<td></td>
		</tr>
		<tr>
			<td>ONPG subtrate</td>
			<td>Pierce</td>
			<td>34055</td>
			<td></td>
		</tr>
		<tr>
			<td>X-gal</td>
			<td>Invitrogen</td>
			<td>15520034</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody anti-B-tubulin</td>
			<td>Sigma-Aldrich</td>
			<td>T8328</td>
			<td>1:2000 dilution</td>
		</tr>
		<tr>
			<td>Antibody anti-peripherin</td>
			<td>Millipore</td>
			<td>AB1530</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr>
			<td>Hoechst dye</td>
			<td>Thermo Fisher</td>
			<td>62249</td>
			<td>1.5 &#181;M final concentration</td>
		</tr>
		<tr>
			<td>Anti-heparan sulfate</td>
			<td>US Biological</td>
			<td>H1890-10</td>
			<td>0.180555556</td>
		</tr>
		<tr>
			<td>Anti gD antibody</td>
			<td>Virostat</td>
			<td>196</td>
			<td>1:10 dilution</td>
		</tr>
		<tr>
			<td>BSA&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A2153-100G</td>
			<td>*Part of SFM media</td>
		</tr>
		<tr>
			<td>BME</td>
			<td>Gibco</td>
			<td>21010-046</td>
			<td>*Part of SFM media</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7021-1KG</td>
			<td>*Part of SFM media</td>
		</tr>
		<tr>
			<td>KIT (Insulin-transferrin-Selenium-A)</td>
			<td>Gibco</td>
			<td>51300-044</td>
			<td>*Part of SFM media</td>
		</tr>
		<tr>
			<td>Vitamin-C</td>
			<td>Sigma-Aldrich</td>
			<td>A4403</td>
			<td>*Part of SFM media</td>
		</tr>
		<tr>
			<td>Putrescine</td>
			<td>Sigma-Aldrich</td>
			<td>P7505</td>
			<td>*Part of SFM media</td>
		</tr>
		<tr>
			<td>488 (goat anti-mouse)</td>
			<td>Life Technologies</td>
			<td>A11029</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy3 (goat anti-rabbit)</td>
			<td>Jackson Immunoresearch laboratories</td>
			<td>111-165-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat serum&#160;</td>
			<td>Vector</td>
			<td>S-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin Solution</td>
			<td>Sigma-Aldrich</td>
			<td>HT5014-120ML</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>10010-031</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X</td>
			<td>Sigma-Aldrich</td>
			<td>T9284-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>VectaShield</td>
			<td>Vector</td>
			<td>H-1500</td>
			<td>Flurescence mount</td>
		</tr>
		<tr>
			<td>Diamond White Glass Coverslides</td>
			<td>Globe Scientific</td>
			<td>1380-20</td>
			<td></td>
		</tr>
	</tbody>
</table>

adult mouse drg explant and dissociated cell models to investigate neuroplasticity and responses to environmental insults including viral infection

This protocol describes an ex vivo model of mouse-derived dorsal root ganglia (DRG) explant and in vitro DRG-derived co-culture of dissociated sensory neurons and glial satellite cells. These are useful and versatile models to investigate a variety of biological responses associated with physiological and pathological conditions of the peripheral nervous system (PNS) ranging from neuron-glial interaction, neuroplasticity, neuroinflammation, and viral infection. The usage of DRG explant is scientifically advantageous compared to simplistic single cells models for multiple reasons. For instance, as an organotypic culture, the DRG explant allows ex vivo transfer of an entire neuronal network including the extracellular microenvironment that play a significant role in all the neuronal and glial functions. Further, DRG explants can also be maintained ex vivo for several days and the culture conditions can be perturbed as desired. In addition, the harvested DRG can be further dissociated into an in vitro co-culture of primary sensory neurons and satellite glial cells to investigate neuronal-glial interaction, neuritogenesis, axonal cone interaction with the extracellular microenvironment, and more general, any aspect associated with the neuronal metabolism. Therefore, the DRG-explant system offers a great deal of flexibility to study a wide array of events related to biological, physiological, and pathological conditions in a cost-effective manner.

Multiple aspects of neuroplasticity and neuron-environment interaction can be investigated using DRG and a single dissociated cell culture model. We began the studies by isolating a DRG explant and DRG-derived dissociated cells as schematically represented in Figure 1. Both tissue and single cells models can be analyzed by using a variety of molecular techniques such as immunofluorescence, Western blot, genomic assays, and other analytical techniques depending on the nature of experimental d...

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Adult Mouse DRG Explant and Dissociated Cell Models to Investigate Neuroplasticity and Responses to Environmental Insults Including Viral Infection

In this report, the advantages of organotypic cultures and dissociated primary cultures of mouse-derived dorsal root ganglia are highlighted to investigate a wide range of mechanisms associated with neuron-glial interaction, neuroplasticity, neuroinflammation, and response to viral infection.

This protocol describes an ex vivo model of mouse-derived dorsal root ganglia (DRG) explant and in vitro DRG-derived co-culture of dissociated sensory ...

The overall goal of this procedure is to create an ex vivo model of dorsal root ganglia explant and/or DRG-derived primary dissociated cell model to investigate neuronal responses to environmental cues. This method can help answer key questions in the field of neuroplasticity, or neuro inflammation in response to pathogens exposure. The main advantage of this technique is that the dorsal root ganglia explants maintains the organ-like complexity useful to start cell cycle into action and changes to intercellular microenvironment due to specific treatments.Watchful demonstration of this method is very critical as in the identification of DRGs as well as harvesting them can be quite difficult due to their size and location. To begin this procedure, expose the vertebral column by cutting down the skin layer of the animal, dorsally using fine scissors. Next, isolate the vertebral column by cutting through the ribs on both sides of the column and through the sacrum, separating the vertebral column from the rest of the animal.Afterward, mount the vertebral column with the ventral side up onto a surgical mat using needles and pins. Then make a double cut on both sides of the vertebral bodies to expose the ventral side of the vertebral canal. Under a surgical microscope gently move the spinal chord on the side to expose the contralateral dorsal spinal roots and to locate the DRG along the dorsal roots of the peripheral nerves.Each ganglion is partially hidden inside the intervertebral foramina. To harvest DRG, pinch the dorsal root with one forceps and gently pull out the DRG from the intervertebral foramen. Then place the second forceps on the spinal nerve peripheral to the DRG and pull the DRG together with the spinal nerve and spinal root.Subsequently, place the collected DRG in a 35 millimeter Petri dish containing three milliliters of ice cold, serum-free media. Transfer each DRG to a dry glass Petri dish. Under a surgical microscope, clean and trim off excess fibers and connective tissue still attached to the DRG using a blade.The DRG is easily identifiable as a bulgy transparent structure along the white spinal nerve, and blood vessels are often found surrounding the DRG. After that place the cleaned DRG in a new Petri dish containing ice cold, serum-free media. Dilute the gelatinous protein mixture in ice cold serum-free media in a 1:1 ratio.Then plate the DRG ex vivo in the 12 well plates pre-coated with 10 or 20 microliters of gelatinous protein mixture. And keep them at 37 degrees Celsius for 30 to 60 minutes. Now gently add 1.5 to two milliliters of serum-free media to the culture system to cover the entire explant and maintain the explants at culturing conditions.Change the DRG growth medium every 72 hours and let the DRG grow for as long as needed. This is a critical step since DRG is anchored to the glass plate using the gelatinous protein mix. Therefore, time oporization and pipetting skills are critical to avoid floating.In this procedure, place all the DRG collected in a 1.5 milliliter sterile tube with F12 media containing Collagenase 4 and incubate it at 37 degrees Celsius for 45 minutes. Afterward, replace the fresh media containing Collagenase 4 and incubate the sample for another 45 minutes. Then treat the explants with two milliliters of F12 media containing 0.025%trypsin at 37 degrees Celsius for 30 minutes immediately after the Collagenase 4 treatment.Incubate them with two milliliters of F12 media containing fetal bovine serum at 37 degrees Celsius for 15 minutes. Afterward, wash the explants three times with two milliliters of F12 media and proceed to mechanically disassociate them with a glass pipette until the media turns cloudy. Following that, filter the dissociated cell culture through a 0.22 micrometer filter to remove any impurities and excess connective tissue.Then centrifuge the filtered cell lysate for two minutes. Remove the supernatant and re-suspend the cell pellet in 500 micro liters of Nurobasal Media. Place the dissociated cells onto laminate-coated cover slides at a preferred cell density.In this step prepare the virus in the correct dilutions in serum-free media to infect the model by using one unit of MOI when working with DRG derived dissociated cells. Place the explants to be infected with virus in a sterile cell plate containing a mixture of serum-free media and virus. Then place the cell plate or tube at 37 degrees Celsius for infection to take place.Now fix the sample in 4%formalin. Wash it three times for 10 minutes each in PBS. Then incubate the sample with the desired primary antibody diluted in PBST and 10%normal goat serum.Store the sample at four degrees Celsius overnight. The next day wash the sample three times for 10 minutes each with PBS. Incubate the sample at room temperature for one hour in the appropriate secondary antibody diluted in PBS.Then wash it three times for 10 minutes each with PBS. Incubate the sample with Hoechst dye in PBS for 20 minutes prior to mounting step. Subsequently mount the sample on glass slides using a fluorescence mounting medium and cover slip.In this image HSV-1 infection of dissociated satellite cells depicted along bata tubulin positive neurites in green is revealed using an antiviral GD antibody in red. Satellite glial cells nuclei are stained blue with Hoechst. The same antibody reveals viral entry in dissociated DRG neurons.Cells are co-labeled with an antibody against Heparan sulfate expressed on the cell membrane. Heparan sulfate is also a component of the extracellular matrix and plays an important role for axonal growth. Neurons are labeled with Pariferin in red while Hoechst blue is used to identify satellite cells nuclei.Once mastered, the ex vivo DRG extractinate can completed in two to two and a half hours if performed properly. While attempting this procedure it is important to remember to respect timing. And not to let the specimens dry out in order to keep the explants alive.Following the harvesting of the DRG, other technique like the dissociation of single cells can be performed in order to answer additional questions such as the effect of environmental ques for the intercellular metabolism. After it's development, this technique paved the way for researchers in the field of neural neurology to explore the neuronal immune response to pathogens. After watching this video, you should have a good understanding on how to generate dorsal root ganglion explants and how to further generate dorsal root ganglion derived primary dissociated cell cultures.Don't forget that working with viruses or any other pathogens can be extremely hazardous and precautions such as via gloves and lab coats, properly dispose your wastes should always be taken while performing this procedure.

adult-mouse-drg-explant-dissociated-cell-models-to-investigate

Research

JoVE Journal

Neuroscience

20.6K visualizações.  Midwestern University. The overall goal of this procedure is to create an ex vivo model of dorsal root ganglia explant and/or DRG-derived primary dissociated cell model to investigate neuronal responses to environmental cues. This method can help answer key questions in the field of neuroplasticity, or neuro inflammation in response to pathogens exposure. The main advantage of this technique is that the dorsal root ganglia explants maintains the organ-like complexity useful to start cell cycle into action and chang...

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