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

All methods described herein that use experimental animals were approved by the Institutional Animal Care and Use Committee (IACUC) of Chang Gung University (CGU 13-014).
1. Collect Lumbar DRG from Experimental Rats
<ol>
	<li>Use 2 to 3 week-old Sprague-Dawley (SD) rats for lumbar DRG collection. 
	NOTE: DRG neurons collected from rats over 4 weeks of age do not grow well under the culture conditions described herein.</li>
	<li>Sterilize all surgical instruments in an autoclave.</li>
	<li>Anesthetize the rat with a 1:1 mixture of tiletamine and zolazepam (20 mg/kg; intraperitoneal injection (IP)) and wait until the animal shows no foot-withdrawal response in a toe-pinch test. 
	NOTE: Different anesthesia strategies can be used successfully in this protocol.</li>
	<li>Sacrifice the rat by decapitation with a commercial guillotine.</li>
	<li>Use the guillotine to isolate the body trunk of the rat between the forelimb and femur. See Figure 1A for a diagram of the region to be collected. 
	NOTE: The caudal cut line should be just rostral to the femur. The lumbar L6 DRG will be excised if the cut site is too high in the spinal column.</li>
	<li>Cut along the sternum and remove all organs/tissues with dissection scissors (Figure 2A-a).</li>
	<li>Cut along the side of trunk to collect the dorsal part of the rat and remove the skin. See Figure 1B for a photograph of the dissected dorsal trunk.</li>
	<li>Prepare the tissue on ice before collecting DRG. Clean the fur and blood from gloves, and sterilize them with 75% ethanol before proceeding to the next step.</li>
	<li>Remove the muscles covering the lumbar spine. First, make two cuts along the sides of the spinal column (left and right) and one lateral cut to mark the rostral extent of the lumbar spine. Then, remove the dorsal muscles of the spine with bone cutting forceps (Figure 2A-b).</li>
	<li>Remove the dorsal portion of the vertebrae with bone cutting forceps and expose the spinal cord.</li>
	<li>Remove the spinal cord with dissection scissors (Figure 2A-c) and forceps (Figure 2A-d).</li>
	<li>Identify the lumbar DRG by counting vertebrae from the last rib (Thoracic Vertebra 13). See Figure 1C for a diagram of the vertebrae positions.</li>
	<li>Collect the bilateral lumbar DRG (L1-L6) with micro-scissors (Figure 2A-f) into a 35-mm culture dish with 2 mL ice-cold serum-free medium. Remove the neuronal fibers (as indicated in Figure 1C) from connecting DRG, then transfer it into the culture dish to improve the purity of the cultures. 
	NOTE: The collected DRG can be kept in medium on ice for about 1 h. Meanwhile, multiple rats can be euthanized to create a larger pool of DRG.</li>
</ol>
2. Primary Culture of Rat Lumber DRG
NOTE: The following steps should be performed in a laminar flow hood.
<ol>
	<li>Prepare culture medium containing 10% fetal bovine serum, 100 mM sodium pyruvate, and 1x penicillin/streptomycin in 1x DMEM-F12.</li>
	<li>Coat the cell-culture treated 24-well plate with 200 &#181;g/mL poly-L-lysine for 2 h then wash with sterilized water.</li>
	<li>Pre-incubate the culture dish with 1 mL culture medium in a 37 &#176;C CO2 incubator before use for least 30 min.</li>
	<li>Transfer the DRG-containing 35 mm dish into a laminar hood, and wash the DRG with serum-free medium 3 times by pipette. 
	NOTE: The outside of the dish should be cleaned with 75% ethanol before transferring into the hood. The 35 mm dish can contain DRG from a number of rats (this will depend on the demands of the experimental design).</li>
	<li>Move the DRG (from a single rat or combined from multiple rats) to a new 35 mm culture dish, which contains 2 mL of collagenase type IA (1 mg/mL in serum-free medium) with sterile tweezers (Figure 2A-e). 
	NOTE: The collagenase solution should be sterilized by passing it through a 0.22 &#181;m syringe filter.</li>
	<li>Digest the DRG in the collagenase solution in a 37 &#176;C CO2 incubator for 30 min.</li>
	<li>Remove the collagenase solution and wash the DRG 3 times in 2 mL Hank&#39;s balanced salt solution (HBSS). 
	NOTE: There may be residual fibers or tissues that come off the DRG into the solution. Remove them by pipette with the washing solution.</li>
	<li>Add 2 mL pre-warmed 0.05% trypsin-EDTA into the DRG-containing 35 mm dish and digest the DRG in a 37 &#176;C CO2 incubator for 30 min.</li>
	<li>Transfer the 2 mL of DRG-containing solution to a 15 mL centrifuge tube by glass pipette. 
	NOTE: The DRG might stick to the glass pipette so this step should be performed with care. DRG loss can be avoided by keeping the DRG-containing solution in the tapered end of a glass pipette (about 0.5 mL) and transferring the solution into the centrifuge tube slowly but without pause.</li>
	<li>Centrifuge the solution at 290 x g for 5 min at 4 &#176;C. Remove the supernatant and add another 2-mL serum-free medium to resuspend the DRG.</li>
	<li>Repeat step 2.10 2 times but change the serum-free medium to pre-warmed culture medium on the last time.</li>
	<li>Manually triturate the DRG approximately 60 times using a flame-polished Pasteur pipette (length 230 mm and tip head inner diameter 1 mm). See Figure 2B for a photograph comparing the orifice of a flame-polished Pasteur pipette to a non-polished pipette. 
	NOTE: The inside diameter of the flame-polished Pasteur pipette is approximately 10% smaller than the control pipette and the inside of the tapered end should be smoother. Be careful not to create bubbles when triturating the cells.</li>
	<li>Remove the poly-L-lysine-coated dish from the CO2 incubator. Aspirate the incubated culture medium from the dish, and seed the dissociated cells onto the coated dish.</li>
	<li>Seed the DRG cells from one rat (bilateral collection from L1-L6, for 12 total DRG) into four wells of a 24-well plate; there are approximately 5 x 104 cells in one well of a 24-well plate. 
	NOTE: This density is suitable for the detection of the released CGRP or SP and also suitable for immunostaining. For Western blot or RNA extraction, seed the DRG cells from one rat (bilateral L1-L6) into one well of a 6-well plate.</li>
	<li>Replace the culture medium on the following day with the addition of 10 &#181;M cytarabine (Ara-C) and 100 ng/mL NGF, and refresh the medium every two days thereafter. 
	NOTE: The thoracic DRG also can also be cultured by this protocol, if they have been collected from the thoracic spine.</li>
</ol>
3. Transfection of NPFFR2 siRNA in DRG Cells
<ol>
	<li>Perform the transfection of NPFFR2 siRNA and control siRNA according the manufacturer&#39;s protocol. 
	NOTE: The protocol will need to be adapted if the chosen transfection reagent is different from the one we used (see the Table of Materials).</li>
	<li>On Day 3 after cell plating, change the medium to 0.5 mL pre-warm serum-free medium and incubate the DRG in a 37 &#176;C CO2 incubator for 1&#160;h.</li>
	<li>Add 50 nM of siRNA (in 1 &#181;L RNase-free water) into 12.5 &#181;L serum-free medium.</li>
	<li>Add 2.5 &#181;L transfection reagent into 10 &#181;L serum-free medium.</li>
	<li>Mix the solution from steps 3.3 and 3.4 by pipette, and incubate this mixed transfection solution for 10 min at room temperature.</li>
	<li>Add the transfection solution into one DRG-containing 24-well plate and mix the solution with medium by gentle shaking. 
	NOTE: Multiple transfection solutions should be deployed at the same time if multiple wells need to be transfected.</li>
	<li>Incubate the DRG in a 37 &#176;C CO2 incubator for 6 h.</li>
	<li>Add 0.5 mL/well of culture medium containing 20% fetal bovine serum, 100 mM sodium pyruvate, and 1x penicillin/streptomycin in 1x DMEM-F12, with the addition of 10 &#181;M Ara-C and 100 ng/mL NGF, into the 24-well plate.</li>
	<li>Incubate the DRG in a 37 &#176;C CO2 incubator for another 66 h (refresh the medium at 48 h).</li>
</ol>
4. Release of Neurotransmitters from Primary DRG Cells
<ol>
	<li>On Day 6 after cells were plated (72 h after siRNA transfection), change the culture medium to 200 &#181;L serum-free medium, and incubate the cells in a 37 &#176;C CO2 incubator for 30 min.</li>
	<li>Add 1 &#181;L stimulation chemical(s) and gently mix the media by pipetting. Incubate the dish in a 37 &#176;C CO2 incubator for the designated time. 
	NOTE: In this article, the cultured cells were stimulated with the NPFFR2 agonist, dNPA (D.Asn-Pro-(N-Me)Ala-Phe-Leu-Phe-Gln-Pro-Gln-Arg- Phe-NH2, 5 nmol), for 1 h.</li>
	<li>Collect the culture medium from the culture dish and centrifuge at 5,000 x g for 5 min at 4 &#176;C to remove any suspended impurities.</li>
	<li>Collect the supernatant from the centrifugation and dilute the samples with phosphate-buffered saline (PBS), as needed. Assay the levels of neurotransmitters with enzyme immunoassay (EIA) kits. 
	NOTE: Here, the supernatants were diluted 1:100 before analyzing the level of CGRP. The supernatant was not diluted before analyzing the level of SP.</li>
</ol>
5. CGRP and SP EIA
<ol>
	<li>Analyze the samples immediately according to the CGRP or SP EIA kit manufacturer&#39;s protocol. 
	NOTE: The protocol will vary depending on the kit used.</li>
	<li>Rinse the CGRP EIA wells 5 times with wash buffer supplied within the kit.</li>
	<li>Add 100 &#181;L samples with 100 &#181;L anti-CGRP acetylcholinesterase (AChE) tracer into the CGRP EIA wells, and add 50 &#181;L samples, 50 &#181;L anti-SP AChE tracer and 50 &#181;L anti-SP antiserum into the SP EIA wells.</li>
	<li>Seal the CGRP and SP wells with plastic film which is supplied within the kits.</li>
	<li>Incubate the wells overnight at 4 &#176;C.</li>
	<li>Wash the wells 5 times with CGRP or SP wash buffer and remove all the residual solution from the wells.</li>
	<li>Add 200 &#181;L Ellman&#39;s reagent into the CGRP or SP wells which is supplied within the corresponding EIA kits.</li>
	<li>Incubate the CGRP wells for 30 min at room temperature, and incubate the SP wells for 90 min at room temperature. Protect the wells from light for both assays.</li>
	<li>Read the plates at wavelength 414 nm and calculate the results according to the corresponding EIA instrument. 
	NOTE: Avoid touching the bottom of the wells by hand all the time and clean the water stains from the well bottom by lens cleaning wipes before adding the Ellman&#39;s reagent.</li>
</ol>

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The authors have nothing to disclose.

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

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

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

dorsal root ganglia isolation and primary culture to study neurotransmitter release

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

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

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

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

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

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

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Research

JoVE Journal

Neuroscience

48.9K Views.  Chang Gung University. 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. 

Video: Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury

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

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

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

Live Imaging of Dorsal Root Axons after Rhizotomy

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

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

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

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

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

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

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

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

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

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

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

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

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors (nAChRs) is an important mechanism for neuromodulation by acetylcholine (ACh). The difficulty with access to probing the signaling mechanisms within intact axons and at nerve terminals both in vitro and in vivo has limited progress in the study of the pre-synaptic components of synaptic plasticity. Here we introduce a gene-chimeric preparation of ventral hippocampal (vHipp)&#8211;accumbens (nAcc) circuit in vitro that allows direct live imaging to analyze both the pre- and post-synaptic components of transmission while selectively varying the genetic profile of the pre- vs post-synaptic neurons. We demonstrate that projections from vHipp microslices, as pre-synaptic axonal input, form multiple, reliable glutamatergic synapses with post-synaptic targets, the dispersed neurons from nAcc. The pre-synaptic localization of various subtypes of nAChRs are detected and the pre-synaptic nicotinic signaling mediated synaptic transmission are monitored by concurrent electrophysiological recording and live cell imaging. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of glutamatergic synaptic plasticity in vitro.

We developed a gene-chimeric preparation of ventral hippocampal &#8211; accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

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

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

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

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

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

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

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

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

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

Laminotomy for Lumbar Dorsal Root Ganglion Access and Injection in Swine

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

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

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

Rapid Isolation of Dorsal Root Ganglion Macrophages

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

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

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