Name: an approach to enhance alignment and myelination of dorsal root ganglion neurons
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Description: Watch this Scientific Journal Video about An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons at JoVE.com

The authors would like to thank Eric Vasco for his assistance in the preparation of the PDMS substrates, Dr. Shiyong Wang in Dr. Marina Mata&#39;s lab at University of Michigan for helpful suggestions and training of the DRG isolation, and Dr. Mark Tuszynski and Dr. W. Marie Campana at UC San Diego for helpful suggestions and protocol for the SC isolation. This study was supported in part by the National Science Foundation (CBET 0941055 and CBET 1510895), the National Institute of Health (R21CA176854, R01GM089866, and R01EB014986).

All procedures for the isolation of the cells were approved by the Institutional Animal Care and Use Committee at Michigan State University.
1. Preparation of Pre-stretched Anisotropic Surface&#160;
<ol>
	<li>Mix a 10:1 solution of base and curing agent and pour the mixture into a tissue culture dish (12 cm diameter). Use 4,900 mg base and 490 mg curing agent for the total crosslinking mixture.</li>
	<li>Keep the gel mixture under vacuum for 20 min to remove air bubbles.</li>
	<li>Place the gel mixture in the oven for overnight curing at 60 &#176;C.</li>
	<li>After the PDMS membrane cures, treat the surface with oxygen plasma using a plasma cleaning/etching system for 3 min at 165 mTorr and 65 sccm flow of O2.</li>
	<li>Cut a piece of rectangular membrane (5&#160;&#215; 3.5 cm) from the dish, while being cognizant which side is oxygen-treated, make sure the oxygen-treated side is facing up and fix onto a stretching frame. Place the frame on a stretching stage and evenly stretch by turning the knob on the stage until it reaches 10% elongation (or some other pre-determined stretch; the elongation can be directly read from the stretching stage) in the longer axis 9. Fix the stretch by tightening the screws on the frame.</li>
	<li>Remove the frame from the stage. Ensure the membrane surface is dry and free of dust, then place a silicone chamber onto the membrane allowing the sticky side of the chamber to attach tightly to the membrane. 
	Note: The silicone chamber provides a well for retaining the cell medium on the PDMS membrane. The device design is shown in Figure 1.</li>
	<li>Sterilize the surface with UV for 10 min.</li>
	<li>Add 1 ml Poly-L-Lysine (PLL) (0.01% in Phosphate Buffered Saline) to the PDMS surface within the chamber, within 6 hr of plasma treatment and incubate for 2 hr at 37 &#176;C prior to seeding the DRG neurons. This enhances cell attachment.</li>
</ol>
2. Isolation of DRG
<ol>
	<li>Prepare standard growth medium for the DRG neurons.</li>
	<li>Prior to the isolation, autoclave the isolation equipment and filter reagents. Add 5 ml of isolation buffer into one well of a 6-well plate, and place the plate on ice. Prepare 10 ml of dissociation medium by adding 1 ml of collagenase A (500 U/ml) to 9 ml of 0.05% Trypsin-EDTA (1 mg/ml), filter the solutions with a 0.22 &#181;m filter and place on ice.</li>
	<li>Use ten to twelve 5 - 7 days&#39; old Sprague-Dawley rats for the isolation. Spray the pups with 75% isopropanol, and sacrifice by decapitation.</li>
	<li>Cut away the skin overlying the spinal cord from the back and remove any excess tissue around the spine. Under a surgical magnifier with the surgical spot lights on, make the first incision from the neck and then one cut along the spine on both sides with scissors. Detach the spine from the body of the pups and remove the excess muscles. Then, use scissors to cut along the long axis of the spine and use tweezers to open the spine completely and extract the spinal cord.Remove the tip from a pipette to create a larger opening for transferring the DRGs with isolation buffer into a sterile 15 ml tube.</li>
	<li>Using a fine pair of tweezers, remove the ganglion from the bone pocket and collect approximately 10 &#8211; 16 DRG from both sides. Trim the nerve roots and transfer the ganglia to the ice-cold isolation buffer.</li>
	<li>Remove the tip from a pipette to create a larger opening for transferring the DRGs with isolation buffer into a sterile 15 ml tube. After the chemical dissociation, centrifuge the ganglia at 900 x g and 4 &#176;C for 5 min.</li>
	<li>Let the tissues settle to the bottom of the tube and gently remove the isolation buffer on top and add 10 ml of dissociation medium into the tube.</li>
	<li>Incubate the dissected tissues in the dissociation medium in a 37 &#176;C water bath for 1 hr while shaking the tube every 5 - 10 min.</li>
	<li>After the chemical dissociation, centrifuge the ganglia at 900 x g and 4 &#176;C for 5 min.</li>
	<li>Remove the supernatant and re-suspend the pellet in 10 ml standard growth media and vortex.</li>
	<li>Centrifuge the dissociated cells as indicated in section 2.9 once again.</li>
	<li>Remove the supernatant, re-suspend the pellet in 12 ml of standard growth media and vortex.</li>
</ol>
3. Culture of DRG on Pre-stretched Surface
<ol>
	<li>Prior to seeding the cells, remove the PLL solutions from the chamber and rinse the PDMS surface with sterile water and air dry.</li>
	<li>After re-suspending the cells, let the suspension set for 1 - 2 min to allow the debris to settle to the bottom of the tube. Add 1.5 ml of cell suspension into each stretch chamber and incubate at 37 &#176;C at 5% CO2.</li>
	<li>To eliminate glial cells, at 1 day in vitro (DIV), add 10 &#181;l (or 15 &#181;l) mixture of fluoro-2 deoxy-uridine and uridine (FDU-U) stock solution (See Table of Materials) to each well. After 7 hr, replace this medium with fresh standard growth medium and place the pre-stretched culture device in a 37 &#176;C incubator with 5% CO2.</li>
	<li>During the cell culture period (2 - 3 weeks), change the media every two days by replacing half the spent medium with fresh medium. Note: After culturing on the stretched and unstretched surfaces for 2 weeks, purified SCs are added to the chamber and cultured for another week (step 4.7).</li>
</ol>
4. Co-culture of Schwann Cells (SCs) with DRG Neurons on Pre-stretched Surface
<ol>
	<li>Isolate SCs as described by Dr. Campana&#39;s group previously 15.
	<ol>
		<li>Briefly, isolate the SCs from a 1 day old pup. Collect and dissociate the sciatic nerves both chemically (Trypsin-EDTA and Collagenase A) and mechanically (18 gauge needle/10 ml syringe) 15.</li>
		<li>Seed the dissociated cells into poly-D-lysine (PDL) coated T25 flask and culture at 37 &#176;C at 5% CO2. On day 5, purify the cells through antibody selection using Anti-thy 1.1 antibody and Rabbit Complement and subculture the purified cells to passage 2 - 4. Use culture medium containing Dulbecco&#39;s Modified Eagle Medium (DMEM), 10% Fetal bovine serum (FBS), 1% Penicillin/Streptomycin (Penn/Strep), 21 &#181;g/ml Bovine Pituitary Extract (BPE), and 4 &#181;M Forskolin.</li>
	</ol>
	</li>
	<li>Remove the culture medium from the SCs, and add 5 ml 0.05% Trypsin-EDTA into the flask. Incubate the cells at 37 &#176;C at 5% CO2 for 2 - 3 min.</li>
	<li>Check cells under the optical microscope using a 10X objective to see if they lift up from the flask, then add 5 ml of culture medium and mix with the cells in the flask.</li>
	<li>Add the cell suspension to a 15 ml centrifuge tube and centrifuge at 200 x g and 20 &#176;C for 5 min.</li>
	<li>Remove the supernatant and resuspend the pellet in 7 ml standard DRG growth media.</li>
	<li>Take 10 &#181;l of cell suspension into a 0.5 ml microcentrifuge tube, mix with 10 &#181;l of trypan blue, and then count the cell number using a hemocytometer under optical microscopy using a 10X objective. Dilute the cell suspension to 5,000 cells per ml by adding DRG growth media.</li>
	<li>Remove 0.5 ml of medium from the DRG culture in the stretched chamber and add 0.5 ml of SC suspension to the DRG culture.</li>
	<li>Culture the cells for 1 week at 37 &#176;C and 5% CO2. Change the media every two days by replacing half the spent medium with fresh standard growth medium for DRG. After 1 week co-culture, process the cells by immunohistochemistry 10.</li>
</ol>

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The authors do not have any conflict of interest to disclose.

To induce axon alignment on pre-stretched surface, there are two critical steps: 1) the PDMS membrane must be flat and of homogenous thickness; and 2) glial cells must be removed from the DRG. After mixing the PDMS and crosslinker and curing in an oven, the crosslinked PDMS gel should be kept on a flat bench top and handled carefully to avoid any tilting. The oxygen plasma treatment of the PDMS membrane should be followed within 6 hr by PLL coating, since the hydrophilicity of the surface (required for cell attachment) a...

In nerve injuries, the proximal and distal nerve stumps are often prevented from direct realignment of nerve fascicles due to the lesion site 1-2. Normally, axon tracts are composed of highly ordered and aligned bundles of axons, which form complex networks of connectivity. However, nerve regeneration is a chaotic process due to poorly organized axon alignment 3-4. Therefore, to generate a sufficient number of regenerating axons that bridge the lesion site, it is necessary to induce well organized axonal alignment. Additionally, demyelination accompanies nerve injuries due to death of the myelinating cells at the injury site. Since demyelination strongly impairs the conductive capacity of surviving axons, treatments targeting demyelination or promoting remyelination are significant for functional recovery after nerve injury 5. Thus the goal of this protocol is to illustrate an engineering approach that addresses these two issues of nerve regeneration.
Surface anisotropy, which is defined as a difference, when measured along different axes, in a material&#39;s physical or mechanical properties, has been applied to influence cell alignment, growth, and migration 6-7. In addition to topography, there are other methods to induce anisotropy. Previously, we investigated surface anisotropy induced by mechanical static pre-stretch of poly-dimethyl-siloxane (PDMS) membrane. The theory of &#34;small deformation super imposed on large&#34; predicted that the effective stiffness the cells sense in the stretched direction differs from the perpendicular direction, and this difference in effective stiffness is due to surface anisotropy 8. Mesenchymal stem cells (MSCs) cultured on a pre-stretched PDMS membrane are able to sense the anisotropy by actively pulling the surface and as a result, align in the pre-stretched direction 9. Similarly, an anisotropic surface arising from a mechanical pre-stretched surface affects the alignment, as well as growth and myelination of dorsal root ganglion (DRG) axons 10. Here we provide a protocol for inducing surface anisotropy on a static pre-stretched PDMS substrate to enhance axon regeneration 10.
To elicit axon alignment, topological features with desired patterns, reported to provide contact guidance through aligned fibers and channels 6,11-12, were demonstrated to facilitate axon alignment 11,13. However, reported techniques for inducing axon alignment through topological features, such as fibers, channels and patterning, were unable to lengthen and increase the thickness of the axons. In contrast, gradual mechanical stretching led to axon alignment in the stretch direction with longer and thicker axons that increased with the magnitude of the stretch 14. However, incorporating a powered motor device in vivo is not feasible. In contrast, static pre-stretched induced anisotropy is less complicated and can be more readily incorporated into future scaffold designs for in vivo applications.
In this protocol, a static pre-stretched cell culture system is used to induce surface anisotropy without topological features. The pre-stretched culture system is composed of a PDMS membrane, a stretchable frame and a stretching stage, whereupon the membrane is fixed onto the frame and a predetermined stretch magnitude is applied on the stretching stage. Freshly isolated DRG neurons cultured on the pre-stretched surface for up to 21 days are monitored for axon alignment and thickness. Subsequently, Schwann cells (SCs) co-cultured with the aligned axons are monitored for myelination. By incorporating pre-stretch induced surface anisotropy we were able to enhance cell alignment-differentiation and axon alignment-growth of MSCs and DRG neurons 9-10, respectively.

<table><tbody><tr><td>Neurobasal Medium 1x</td><td>GibcoBRL</td><td>21103-049</td><td></td></tr><tr><td>B27 Supplement 50x</td><td>GibcoBRL</td><td>17504-044</td><td></td></tr><tr><td>Glutamax-I 100x</td><td>GibcoBRL</td><td>35050-061</td><td></td></tr><tr><td>Albumax-I</td><td>GibcoBRL</td><td>11020-021</td><td></td></tr><tr><td>Nerve Growth Factor-7S</td><td>Invitrogen</td><td>13290-010</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>GibcoBRL</td><td>15140-122</td><td></td></tr><tr><td>0.05% Trypsin-EDTA/1 mM EDTA</td><td>GibcoBRL</td><td>25300-054</td><td></td></tr><tr><td>Poly-L-Lysine</td><td>Trevigen</td><td>&lt;a href=&quot;http://www.trevigen.com/item/2/7/64/220/Cultrex_PolyLLysine/&quot;&gt;3438-100-01&lt;/a&gt;</td><td></td></tr><tr><td>Poly-D-Lysine</td><td>Sigma</td><td>p-6407</td><td></td></tr><tr><td>Fluoro-2 deoxy-uridine</td><td>Sigma</td><td>F0503</td><td></td></tr><tr><td>Uridine</td><td>Sigma</td><td>U3003</td><td></td></tr><tr><td>Hank&amp;rsquo;s Balanced Salt Solution (HBSS)</td><td>Invitrogen</td><td>14170-112</td><td>Isolation Buffer</td></tr><tr><td>Type I Collagenase</td><td>Worthington</td><td>LS004196</td><td></td></tr><tr><td>DMEM</td><td>Gibco</td><td>11885</td><td></td></tr><tr><td>Heat inactivated Fetal Bovine Serum</td><td>Hyclone</td><td>SH30080.03</td><td></td></tr><tr><td>BPE</td><td>Clonetics</td><td>CC-4009</td><td></td></tr><tr><td>Forskolin</td><td>Calbiochem</td><td>344270</td><td></td></tr><tr><td>Silicone chamber</td><td>Greiner bio-one</td><td>FlexiPERM ConA</td><td></td></tr><tr><td>Plasma cleaning/etching system</td><td>March Instruments</td><td>PX-250</td><td></td></tr><tr><td>Anti-Thy 1.1 antibody</td><td>Sigma- Aldrich</td><td>M7898</td><td></td></tr><tr><td>Rabbit Complement</td><td>Sigma- Aldrich</td><td>S-7764</td><td></td></tr><tr><td>Standard growth medium</td><td></td><td></td><td>For 500 ml Neurobasal Medium 1x, add 10 ml of B-27 50x, 5 ml of Glutamax-I 100x, 2.5 ml of Penicillin/Streptomycin (Penn/Strep), 1 ml of Albumax-I, and 1 &amp;mu;l of NGF-- 7S (50 &amp;mu;g/ml).</td></tr><tr><td>FDU Uridine stock solution</td><td></td><td></td><td>FDU 100 mg in 10 ml of ddH&lt;sub&gt;2&lt;/sub&gt;O (10 mg/ml), filter in the hood and divided in 500 &amp;mu;l aliquots and store at -20 &amp;ordm;C. Uridine 5 g in 166.7 ml of ddH&lt;sub&gt;2&lt;/sub&gt;O (33 mg/ml), filter in hood, divide in 200 &amp;mu;l aliquots and store at -20 &amp;ordm;C.
Take 61.5 &amp;mu;l of FDU (10 mg/ml) and 20.5 &amp;mu;l of Uridine(33 mg/ml), and add 4,918 &amp;mu;l of ddH&lt;sub&gt;2&lt;/sub&gt;O to a final stock concentration, then divide in 1 ml aliquots and store at -20 &amp;ordm;C.</td></tr></tbody></table>

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.

The pre-stretched cell culture system promoted DRG axon alignment 10. DRG neurons were cultured onto pre-stretched and unstretched surfaces for 12 days. The axons were stained for &#946;-III-tubulin to demonstrate their alignment. Figure 2 compares axon orientation on the pre-stretched and unstretched PDMS substrates after 12 days of culture. The DRG axons aligned parallel to the stretched direction, whereas they showed random alignment and formed an interconne...

Watch this Scientific Journal Video about An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons at JoVE.com

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

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 ...

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8.4K Views.  Michigan State University. 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.

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

Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for neuronal survival and function. Neurotrophins are specifically required to maintain the survival and differentiation of innervating sensory neurons but the question of how these target-derived neurotrophins communicate to the cell body of innervating neurons has been an area of active research for over 30 years. The most commonly accepted model of how neurotrophin signals reach the cell body proposes that signaling endosomes carry this signal retrogradely along the axon. In order to study retrograde transport, a culture system was originally devised by Robert Campenot, in which cell bodies are isolated from their axons. The technique of preparing these compartmented chambers for culturing sensory neurons recapitulates the selective stimulation of neuron terminals that occurs in vivo following release of target-derived neurotrophins. Retrograde signaling events that require long-range microtubule dependent retrograde transport have important implications for the treatment of neurodegenerative disorders.

Here we describe the technique of preparing and maintaining compartmented chambers for culturing sensory neurons of the dorsal root ganglia.

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for ...

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 ...

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 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 ...

Use of In Vivo Single-fiber Recording and Intact Dorsal Root Ganglion with Attached Sciatic Nerve to Examine the Mechanism of Conduction Failure

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for nerve fibers in the central and peripheral nervous systems. This method is particularly applicable to dorsal root ganglia (DRG), which are primary sensory neurons that exhibit a pseudo-unipolar structure of nervous processes. The patterns and features of the action potentials passed along axons are recordable in these neurons. The present study uses in vivo single-fiber recordings to observe the conduction failure of sciatic nerves in complete Freund&#8217;s adjuvant (CFA)-treated rats. As the underlying mechanism cannot be studied using in vivo single-fiber recordings, patch-clamp-recordings of DRG neurons are performed on preparations of intact DRG with the attached sciatic nerve. These recordings reveal a positive correlation between conduction failure and the rising slope of the after-hyperpolarization potential (AHP) of DRG neurons in CFA-treated animals. The protocol for in vivo single fiber-recordings allows the classification of nerve fibers via the measurement of conduction velocity and monitoring of abnormal conditions in nerve fibers in certain diseases. Intact DRG with attached peripheral nerve allows observation of the activity of DRG neurons in most physiological conditions. Conclusively, single-fiber recording combined with electrophysiological recording of intact DRGs is an effective method to examine the role of conduction failure during the analgesic process.

Single-fiber recording is an effective electrophysiological technique that is applicable to the central and peripheral nervous systems. Along with the preparation of intact DRG with the attached sciatic nerve, the mechanism of conduction failure is examined. Both protocols improve the understanding of the peripheral nervous system&#39;s relationship with pain.

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for ...

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

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

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

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

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

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 ...

Neuronal Enrichment of Dorsal Root Ganglia by Immunopanning

Source: Maguire, A. D., et al. <a href="https://app.jove.com/v/64603">Enrichment of Adult Mouse Dorsal Root Ganglia Neuron Cultures by Immunopanning.</a> J. Vis. Exp. (2023)
This video demonstrates a protocol for enriching neuronal cultures from dorsal root ganglia by immunopanning, thereby improving their purity and utility for neurological research.

1. Experimental method

	Day 1: preparation of dishes
	
		In a biosafety cabinet, coat the desired culture surface with enough poly-D-lysine to cover the ...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Co-culture and Interaction Studies

Co-Culture of Schwann Cells with DRG Neurons on a Pre-Stretched Membrane

Source: Liu, C., et al. <a href="https://app.jove.com/v/54085">An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons.</a> J. Vis. Exp. (2016).
The video demonstrates the co-culturing of dorsal root ganglion (DRG) neurons with Schwann cells. DRG neurons are first grown on a pre-stretched membrane for aligned axon growth. Schwann cells are then introduced, which attach to the axons, insulating them and promoting neural cell survival and function.

1. Culture of dorsal root ganglion (DRG) on Pre-stretched Surface

	Preparation of Pre-stretched Anisotropic Surface
	
		Mix a 10:1 solution of base and ...

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

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