Name: adult mouse drg explant and dissociated cell models to investigate neuroplasticity and responses to environmental insults including viral infection
Uploaded: 2018-03-09T13:30:00.000+00:00
Duration: 9 min 23 s
Description: Watch this Scientific Journal Video about Adult Mouse DRG Explant and Dissociated Cell Models to Investigate Neuroplasticity and Responses to Environmental Insults Including Viral Infection at JoVE.co

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 column by cutting through the ribs on either side of the column and through the sacrum separating the vertebral column from the rest of the animal.</li>
		<li>Mount the vertebral column (ventral side up) onto a surgical mat using needles/pins.</li>
	</ol>
	</li>
	<li>Using fine scissors make a double cut on both sides of the vertebral bodies to expose the ventral side of the vertebral canal.</li>
	<li>Under a surgical microscope (magnification set to 4X), use scissors to gently move the spinal cord 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.</li>
	<li>To harvest the DRG, pinch the dorsal root (between the spinal cord and the DRG) with one forceps and gently pull out the DRG from the intervertebral foramen.</li>
	<li>Place the second forceps on the spinal nerve peripheral to the DRG and pull the DRG together with the spinal nerve and spinal root.</li>
	<li>Place the collected DRG in a 35 mm Petri dish containing 3 mL of ice-cold serum free media (SFM)34.</li>
	<li>Transfer each individual DRG in a dry glass Petri dish and, 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/root. Blood vessels often are found surrounding the DRG.</li>
	<li>Place the cleaned DRG in a new Petri dish containing ice-cold SFM media.</li>
	<li>Dilute the gelatinous protein mixture (see Table of Materials) in ice-cold SFM (1:1).</li>
	<li>Plate the DRG ex vivo in 12-well plates pre-coated with 10/20 &#181;L of gelatinous protein mixture and set them inside the incubator at 37 &#176;C and 5% CO2 for 30-60 min.</li>
	<li>Gently add 1.5-2 mL of SFM to the culture system to cover the entire explant and maintain the explants at culturing conditions (37 &#176;C and 5% CO2). 
	NOTE: This is a critical step because the DRG is anchored to the glass dishes only by the polymerized gelatinous protein mixture. Time of polymerization and pipetting skills are critical to avoid floating.</li>
	<li>Change the medium of growing DRG every 72 h and let the DRG grow for as long as needed.</li>
</ol>
2. Isolating Single Cell Neurons from DRG
<ol>
	<li>Place all the DRG collected in a 1.5 mL sterile tube with 1.2 mL of F12 media containing 1.25 mg/mL of collagenase IV and incubate it at 37 &#176;C and 5% CO2 for 45 min. Repeat this step for another 45 min after the first incubation.</li>
	<li>Treat explants with 2 mL of F12 media containing trypsin (0.025%) for 30 min immediately after the collagenase IV treatment at 37 &#176;C and 5% CO2.</li>
	<li>Incubate with 2 mL of F12 media containing fetal bovine serum (FBS; 33%) at 37 &#176;C and 5% CO2 for 15 min.</li>
	<li>Wash explants three times with 2 mL of F12 media and proceed to mechanically dissociate them with a glass pipette until the media turns cloudy. 
	NOTE: This procedure involves collection of clean/trimmed DRG from the animal as explained in the prior section. When performing mechanical dissociation of explants, be gentle and do not use excessive force since it can lead to spillage and loss of working material.</li>
	<li>Filter the dissociated cell culture through a 0.22 &#181;m filter to remove any impurities and excess connective tissue. Centrifuge the filtered cell lysate at (2,500 x g) for 2 min.</li>
	<li>Remove the supernatant and resuspend the cell pellet in 500 &#181;L of neurobasal media containing supplement for neuronal culture (1x), antibiotic mixture (1x), L-glutamate (0.5 mM), and nerve growth factor (5 &#181;g/mL) (see Table of Materials).</li>
	<li>Plate the dissociated cells onto laminin-coated cover slides (50 &#181;g/mL; see Table of Materials) at a preferred cell density. Determine the cell density using a cell counter. 
	NOTE: We plated cells at 25,000 cells/cover slide. This technique allows dissociation of both neurons and glial satellite cells. The glial component co-cultured with the pseudounipolar neurons plays a critical role for neuronal survival.</li>
</ol>
3. HSV-1 Infection of DRG Explants and DRG-derived Dissociated Cells
NOTE: This work was done by strictly following the biosafety level-2 (BSL-2) requirements to which we have a fully equipped lab that is approved by the Midwestern University biosafety committee. A KOS strain of HSV-1 was used in this study. Please take appropriate measurements and safety precautions as per local institutions guidelines if working with virus strains.
<ol>
	<li>Determine and prepare the virus in the correct dilutions in SFM media to infect the model. The virus used in this study was KOS strain of HSV-1. When working with DRG-derived dissociated cells, use 1 unit of multiplicity of infection (MOI) for infection, which means the number of virus equal the number of cells.</li>
	<li>If infecting DRG explants, use the number of virions (e.g., 10,000 virions) because an exact number of cells in an explant cannot be determined.</li>
	<li>Place the explant/cells to be infected with virus in a sterile cell plate or tube containing a mixture of SFM media and virus. 
	NOTE: We used 25,000 virions for a cover slide containing 25,000 cells (1 MOI). To infect an explant, we use 10,000 virions.</li>
	<li>Place the cell plate or tube at 37 &#176;C for infection to take place; time of viral exposure may vary depending on viral infectivity. 
	NOTE: Viral entry was confirmed by using ortho-nitrophenyl-&#946;-galactoside (ONPG) and 5-bromo-4-chloro-3-indolyl-D-galactopyranoside (X-gal) assays26.</li>
</ol>
4. Immunofluorescence
<ol>
	<li>Fix the explants and single cell samples in 4% formalin prepared in phosphate buffered saline (PBS). Wash samples 3 times for 10 min each in PBS.</li>
	<li>Incubate the samples with desired primary antibodies (anti-&#946;-tubulin, anti-peripherin, anti-heparan sulfate (HS), and/or anti-glycoprotein D (gD) antibody; see Table of Materials for dilutions) diluted in PBS buffer with 0.3% Triton-X (PBST) and 10% normal goat serum. Store samples at 4 &#176;C overnight.</li>
	<li>Wash the samples 3 times for 10 min each with PBS. Incubate samples at room temperature for 1 h in the appropriate secondary antibody (488 and/or Cy3; see Table of Materials for dilutions) diluted in PBS.</li>
	<li>Wash the samples 3 times for 10 min each with PBS. Incubate the samples with Hoechst dye (1.5 &#181;M) in PBS for 20 min prior to mounting step.</li>
	<li>Mount the samples on glass slides using a fluorescence mounting medium and coverslip.</li>
</ol>

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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 small molecules in DRG explants could exploit this explant model for drug development. In vitro models, especially single-cell systems, are often too simplistic to correlate to physiologically relevant events; while in vivo models are challenging to precisely manipulate, partially due to the immune response to injury, glial scarring, and the complexity of the surrounding ECM along with the financial expense. In contrast, the DRG explant is an organotypic culture that allows ex vivo transfer of an entire organ with the complexity of a neuronal network, including the extracellular microenvironment that plays a significant role in all the neuronal and glial functions. However, the generation of an ex vivo model can only resemble but not fully reciprocate all the conditions found in vivo. Two important limitations to keep in mind when using this model are the timing and the absence of systemic feedbacks, which can only truly be represented in vivo. The limited time that the DRG can be maintained vital ex vivo makes this model more suitable for investigation of acute conditions and thus not a very reliable model for chronic conditions. Similarly, the ex vivo model is not ideal for studies that may be effected by the absence of responses from other organs or systems (e.g., the immune system).
We were able to successfully isolate the explant ex vivo DRG from both adult and embryo mice and rats and use this model to investigate HSV-1 infection as well as multiple other aspects of neuroplasticity and its effects on translational medicine. In order to allow sensory fibers to grow outside the explant, the connective surrounding capsule was partially removed1.
To be considered, the gelatinous protein mixture extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcom isrich in ECM proteins such as laminin, collagen IV, heparin sulfate proteoglycans (HSPG), and a number of growth factors38. In this protocol, the gelatinous protein mixture is diluted 1:1 with culture medium and allowed to polymerize for 30-60 min at 37 &#176;C before adding the final volume of medium. The gelatinous protein mixture polymerizes at room temperature; therefore, it is suggested to refrigerate the mixture until explants are plated and to use cold pipette tips to distribute 10-20 &#181;L drops onto the 12-well dishes. Timing is critical for the final efficiency of the culture procedure and may vary depending on the type (and manufacture) of the gelatinous protein mixture used. If the gelatinous protein mixture is not fully polymerized or it is already solidified, the explant can float or dry out leading to unsuccessful growth. We encourage investigators to empirically determine the appropriate temperature, time, and gelatinous protein mixture dilution for a successful model. We reached a 55-70% efficiency of successful growth of DRG explants maintained ex vivo for up to 7-10 days. The explant can be further dissociated into DRG-derived primary sensory neurons and satellite cells co-culture.

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><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&lt;br/&gt; 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 &amp;micro;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&amp;nbsp;</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&amp;nbsp;</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 design and aims. First, we used our DRG explant model to investigate the effect of biochemical cues on axonal growth (Figure 2). In double or triple immunofluorescence, the differential expression of peripherin (Figure 2A, B; red) and &#946;-tubulin (Figure 2A, B; green) were confirmed both within the neuronal cell bodies and the peripheral neurite sprouts. Cell nuclei were identified with Hoechst stain (Figure 2A; blue). The expression of the above-mentioned markers was further analyzed in the entire DRG explant after fixation in 4% paraformaldehyde and microtome-sectioning (Figure 3A). Our results confirmed that peripherin and &#946;-tubulin are selectively expressed in two subpopulations of ganglionic neurons with peripherin being expressed in the &#34;small, light&#34; and &#946;-tubulin in the &#34;large, dark&#34; subpopulations of neurons, respectively. The neurites immunolabeled with either marker emerging from the sensory neurons were found coursing through the entire ganglion before exiting into the periphery. Hoechst stain mostly identified the position of the glial satellite nuclei within the ganglion. As indicated in Figure 3B, the neuron-satellite cell interaction can be better visualized after the full cell dissociation technique. Blue-stained satellite cell nuclei were seen surrounding a &#946;-tubulin-positive sensory neuronal soma and its extensive neurites.
We further utilized the DRG-derived dissociated cells to investigate HSV-1 infection and associated inflammatory response within the sensory neurons (Figure 4). After only 1 h of post infection (p.i.), HSV-1 entry was detected by using anti-HSV-1 gD antibody both in glial cells (Figure 4A) and DRG neurons (Figure 4B). In addition, HS staining was further carried out since HS provides a docking sites for virus attachment or binding to host cell. HS has been known as a critical component of the multi-functional extracellular matrix (ECM) and extensively documented to play a major role in cell adhesion, cell-to-cell signaling, and ECM remodeling during wound healing, embryonic development, cancer invasion, fibrosis, and in neuroinflammation35,36. Interestingly, we observed HS staining in the ECM suggesting its potential role in the microenvironment that allows for axonal growth (Figure 4C). Finally, for &#946;-tubulin staining we used as a positive marker for the neurites to confirm the integrity of the neurons.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56757/56757fig1.jpg" /> 
Figure 1. Schematic representation of the experimental DRG and single cell culture models. DRG were isolated from Adult NIH/SWISS mice, cleaned from excessive capsular connective tissue, and either explanted ex vivo as a whole explant or dissociated into primary co-cultures of sensory neurons and satellite cells. Both models can be used for further analyses with multiple analytical techniques. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56757/56757fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56757/56757fig2.jpg" /> 
Figure 2. Development of DRG organotypic culture as a model system to investigate axonal growth. DRG grown for 6 days ex vivo were used to investigate axonal sprouting from the explant. We show by immunofluorescence how different biochemical conditions can affect the distribution of fiber sprouts: growing randomly and disorganized ( A) compared to a more linear and organized fashion ( B). The explants are labeled with anti-&#946;-Tubulin (green) and anti-peripherin (red). Cell nuclei are stained with Hoechst stain (blue). Scale bars = 100 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56757/56757fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56757/56757fig3.jpg" /> 
Figure 3. Molecular characterization of the isolated neuronal culture model. Sections of the whole DRG were immunolabeled with antibodies against peripherin and &#946;-tubulin showing selective expression of the two markers in two subpopulations of ganglionic neurons. Peripherin (red) is expressed in the &#34;small, light&#34; neurons and &#946;-tubulin (green) is expressed in the &#34;large, dark&#34; neurons. Hoechst stain mostly identified the position of the glial satellite nuclei within the ganglion ( A). The neuron-satellite cell interaction can be better visualized after full cell dissociation technique ( B). Here we show a &#946;-tubulin-positive sensory neuronal body and its neurites surrounded by satellite glial cells (Hoechst: nuclei, blue). Scale bars, A = 100 &#181;m; B = 25 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56757/56757fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56757/56757fig4.jpg" /> 
Figure 4. Investigation of HSV-1 entry model in DRG-derived dissociated cells. HSV-1 infection of dissociated satellite cells depicted along &#946;-tubulin-positive neurites (green) is revealed using an anti-viral gD antibody (red). Satellite glial cells nuclei are stained blue with Hoechst ( A). The same antibody reveals viral entry in dissociated DRG neurons ( B). Cells are co-labeled with an antibody against heparan sulfate (HS, green) expressed on the cell membrane. HS is also a component of the extracellular matrix (green) and plays an important role for axonal growth ( C). Neurons are labeled with peripherin (red) while Hoechst blue is used to identify satellite cell&#39;s nuclei. Scale bars = 25 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56757/56757fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Adult Mouse DRG Explant and Dissociated Cell Models to Investigate Neuroplasticity and Responses to Environmental Insults Including Viral Infection at JoVE.co

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Neuroscience

21.2K Views.  Midwestern University. In dit verslag zijn de voordelen van organotypic culturen en gedissocieerde primaire culturen van muis-afgeleide dorsal root ganglia gemarkeerd om te onderzoeken van een breed scala van mechanismen die zijn gekoppeld aan neuron-gliale interactie, Neuroplasticiteit, neuroinflammation, en reactie op virale infectie.

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

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Myelination is a complex process that involves both neurons and the myelin forming glial cells, oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS). We use an in vitro myelination assay, an established model for studying CNS myelination in vitro. To do this, oligodendrocyte precursor cells (OPCs) are added to the purified primary rodent dorsal root ganglion (DRG) neurons to form myelinating co-cultures. In order to specifically interrogate the roles that particular proteins expressed by oligodendrocytes exert upon myelination we have developed protocols that selectively transduce OPCs using the lentivirus overexpressing wild type, constitutively active or dominant negative proteins before being seeded onto the DRG neurons. This allows us to specifically interrogate the roles of these oligodendroglial proteins in regulating myelination. The protocols can also be applied in the study of other cell types, thus providing an approach that allows selective manipulation of proteins expressed by a desired cell type, such as oligodendrocytes for the targeted study of signaling and compensation mechanisms. In conclusion, combining the in vitro myelination assay with lentiviral infected OPCs provides a strategic tool for the analysis of molecular mechanisms involved in myelination.

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Here we present protocols that offer a flexible and strategic foundation for virally manipulating oligodendrocyte precursor cells to overexpress proteins of interest in order to specifically interrogate their role in oligodendrocytes via the in vitro model of central nervous system myelination.

Myelination is a complex process that involves both neurons and the myelin forming glial cells, oligodendrocytes in the central nervous system (CNS) and ...

Developmental Biology

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

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

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

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

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

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

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

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

Dorsal Root 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 ...

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 ...

Mouse Footpad Inoculation Model to Study Viral-Induced Neuroinflammatory Responses

This protocol describes a footpad inoculation model used to study the initiation and development of neuroinflammatory responses during alphaherpesvirus infection in mice. As alphaherpesviruses are main invaders of the peripheral nervous system (PNS), this model is suitable to characterize the kinetics of viral replication, its spread from the PNS to CNS, and associated neuroinflammatory responses. The footpad inoculation model allows virus particles to spread from a primary infection site in the footpad epidermis to sensory and sympathetic nerve fibers that innervate the epidermis, sweat glands, and dermis. The infection spreads via the sciatic nerve to the dorsal root ganglia (DRG) and ultimately through the spinal cord to the brain. Here, a mouse footpad is inoculated with pseudorabies virus (PRV), an alphaherpesvirus closely related to herpes simplex virus (HSV) and varicella-zoster virus (VZV). This model demonstrates that PRV infection induces severe inflammation, characterized by neutrophil infiltration in the footpad and DRG. High concentrations of inflammatory cytokines are subsequently detected in homogenized tissues by ELISA. In addition, a strong correlation is observed between PRV gene and protein expression (via qPCR and IF staining) in DRG and the production of pro-inflammatory cytokines. Therefore, the footpad inoculation model provides a better understanding of the processes underlying alphaherpesvirus-induced neuropathies and may lead to the development of innovative therapeutic strategies. In addition, the model can guide research on peripheral neuropathies, such as multiple sclerosis and associated viral-induced damage to the PNS. Ultimately, it can serve as a cost-effective in vivo tool for drug development.

The footpad inoculation model is a valuable tool for characterizing viral-induced neuroinflammatory responses in vivo. In particular, it provides a clear assessment of viral kinetics and associated immunopathological processes initiated in the peripheral nervous system.

This protocol describes a footpad inoculation model used to study the initiation and development of neuroinflammatory responses during alphaherpesvirus ...

Immunology and Infection

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

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory bulb. Throughout adult life, they are key for axonal growing of newly generated olfactory neurons, from the lamina propria to the ONL. Due to their pro-regenerative properties, these cells have been used to foster axonal regeneration in spinal cord or optic nerve injury models.
We present an in vitro model to assay and measure OEG neuroregenerative capacity after neural injury. In this model, reversibly immortalized human OEG (ihOEG) is cultured as a monolayer, retinas are extracted from adult rats and retinal ganglion neurons (RGN) are cocultured onto the OEG monolayer. After 96 h, axonal and somatodendritic markers in RGNs are analyzed by immunofluorescence and the number of RGNs with axon and the mean axonal length/neuron are quantified.
This protocol has the advantage over other in vitro assays that rely on embryonic or postnatal neurons, that it evaluates OEG neuroregenerative properties in adult tissue. Also, it is not only useful for assessing the neuroregenerative potential of ihOEG but can be extended to different sources of OEG or other glial cells.

We present an in vitro model to assess olfactory ensheathing glia (OEG) neuroregenerative capacity, after neural injury. It is based on a coculture of axotomized adult retinal ganglion neurons (RGN) on OEG monolayers and subsequent study of axonal regeneration, by analyzing RGN axonal and somatodendritic markers.

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory ...

Advanced In Vitro Model for Studying Neuronal Hypersensitivity in DRG Cultures

Harvesting, Embedding, and Culturing Dorsal Root Ganglia in Multi-compartment Devices to Study Peripheral Neuronal Features

The most common peripheral neuronal feature of pain is a lowered stimulation threshold or hypersensitivity of terminal nerves from the dorsal root ganglia (DRG). One proposed cause of this hypersensitivity is associated with the interaction between immune cells in the peripheral tissue and neurons. In vitro models have provided foundational knowledge in understanding how these mechanisms result in nociceptor hypersensitivity. However, in vitro models face the challenge of translating efficacy to humans. To address this challenge, a physiologically and anatomically relevant in vitro model has been developed for the culture of intact dorsal root ganglia (DRGs) in three isolated compartments in a 48-well plate. Primary DRGs are harvested from adult Sprague Dawley rats after humane euthanasia. Excess nerve roots are trimmed, and the DRG is cut into appropriate sizes for culture. DRGs are then grown in natural hydrogels, enabling robust growth in all compartments. This multi-compartment system offers anatomically relevant isolation of the DRG cell bodies from neurites, physiologically relevant cell types, and mechanical properties to study the interactions between neural and immune cells. Thus, this culture platform provides a valuable tool for investigating treatment isolation strategies, ultimately leading to an improved screening approach for predicting pain.

Author Spotlight: In Vitro and In Vivo Models to Enhance Chronic Pain Treatment Efficacy

This protocol provides a technique to harvest and culture explanted dorsal root ganglion (DRG) from adult Sprague Dawley rats in a multi-compartment (MC) device.

The most common peripheral neuronal feature of pain is a lowered stimulation threshold or hypersensitivity of terminal nerves from the dorsal root ganglia ...

Bioengineering

Long-Term Engraftment of h-SC-NES Cells in Spinal Cord Organotypic Cultures

Long-Term Mouse Spinal Cord Organotypic Slice Culture as a Platform for Validating Cell Transplantation in Spinal Cord Injury

Resolutive cures for spinal cord injuries (SCIs) are still lacking, due to the complex pathophysiology. One of the most promising regenerative approaches is based on stem cell transplantation to replace lost tissue and promote functional recovery. This approach should be further explored better in vitro and ex vivo for safety and efficacy before proceeding with more expensive and time-consuming animal testing. In this work, we show the establishment of a long-term platform based on mouse spinal cord (SC) organotypic slices transplanted with human neural stem cells to test cellular replacement therapies for SCIs.
Standard SC organotypic cultures are maintained for around 2 or 3 weeks in vitro. Here, we describe an optimized protocol for long-term maintenance (&#8805;30 days) for up to 90 days. The medium used for long-term culturing of SC slices was also optimized for transplanting neural stem cells into the organotypic model. Human SC-derived neuroepithelial stem (h-SC-NES) cells carrying a green fluorescent protein (GFP) reporter were transplanted into mouse SC slices. Thirty days after the transplant, cells still show GFP expression and a low apoptotic rate, suggesting that the optimized environment sustained their survival and integration inside the tissue. This protocol represents a robust reference for efficiently testing cell replacement therapies in the SC tissue. This platform will allow researchers to perform an ex vivo pre-screening of different cell transplantation therapies, helping them to choose the most appropriate strategy before proceeding with in vivo experiments.

Author Spotlight: Long-Term Spinal Cord Slice Culture for Advancing Spinal Cord Regeneration Therapies

In this paper, we provide a reproducible method to generate and maintain long-term spinal cord organotypic slices transplanted with neural stem cells as an ex vivo model for testing cellular replacement therapies.

Resolutive cures for spinal cord injuries (SCIs) are still lacking, due to the complex pathophysiology. One of the most promising regenerative approaches ...

AAV Systems and Mouse Models to Study Ectopic Neurod1 Expression Post-Stroke

AAV Systems and Mouse Models for Investigating Ectopic Expression of Neurod1 in Transduced Cells at Subacute and Chronic Times Post-Ischemic Stroke

Ectopic expression of neurogenic factors in vivo has emerged as a promising approach for replacing lost neurons in disease models. The use of neural basic helix-loop-helix (bHLH) transcription factors via non-propagating virus-like particle systems, including retrovirus, lentivirus, and adeno-associated virus (AAV), has been extensively reported. For in vivo experiments, AAVs are increasingly used due to their low pathogenicity and potential for translatability. This protocol describes two AAV systems for investigating the ectopic expression of transcription factors in transduced cells post-ischemic stroke. In both systems, Neurod1 expression is controlled by the short GFAP (gfaABC(1)D) promoter, which is upregulated in reactive astrocytes post-stroke as well as in endogenous neurons when combined with neurogenic factor expression. In the ischemic stroke model described, focal ischemia is induced by injecting endothelin-1 (ET-1) into the motor cortex of mice, creating a lesion surrounded by reactive GFAP-expressing astrocytes and surviving neurons. Intracerebral injections of AAV are performed to ectopically induce the expression of Neurod1 in the subacute (7 days) and chronic (21 days) phases post-stroke. Within weeks following AAV injection, a significantly higher number of neurons among transduced cells are identified in mice ectopically expressing Neurod1 compared to mice receiving AAV control viruses. The AAV-based strategies used replicated observed outcomes of increased numbers of neurons expressing the reporter gene in a model of mild-to-moderate cortical stroke. This protocol establishes a standard platform for exploring the effects of ectopic expression of transcription factors delivered with AAV-based systems, contributing to the understanding of neurogenic factor expression in the context of stroke.

This protocol describes the viral-mediated ectopic expression of Neurod1 following cortical ischemic stroke. Neurod1 is delivered (1) using the Cre-Flex AAV system in wild-type mice during the subacute phase post-stroke (7 days) and (2) using a single AAV vector in conditional reporter mice during the chronic phase post-stroke (21 days).

Ectopic expression of neurogenic factors in vivo has emerged as a promising approach for replacing lost neurons in disease models. The use of neural basic ...

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

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