The study was supported by: Foundation for Anesthesia Education and Research (XY); the UCSF Department of Anesthesia and Perioperative Care (XY); and 1R01NS100801-01(GZ). This study was supported in part by HDFCCC Laboratory for Cell Analysis Shared Resources Facility through a grant from the NIH (P30CA082103).

All animal experiments were approved by the Institutional Animal Care and Use Committee at the University of California San Francisco and were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals.
1. Collect lumbar DRG from experimental mice
<ol>
	<li>Before starting the experiment, prepare the working solution of the density gradient medium (e.g., Percoll) by mixing 9 volumes of the medium with 1 volume of Ca++/Mg++-free 10x HBSS. Keep it on ice.</li>
	<li>Anesthetize the mouse with 2.5% Avertin. Confirm that the animal is fully anesthetized by the lack of response to the hind paw pinch. 
	NOTE: Both male and female mice aged 6-8 weeks were used.</li>
	<li>Perfuse the mouse transcardially with 10 mL of pre-chilled 1x PBS.
	<ol>
		<li>Place the mouse in the supine position with four paws secured with the tape inside a chemical fume hood. Lift the skin below the rib cage by the forceps, and make a small incision with surgical scissors to expose the liver and diaphragm.</li>
		<li>Continue to use scissors to cut the diaphragm and the rib cage, open the pleural cavity to expose the beating heart.</li>
		<li>Quickly cut the right atrial appendage with iris scissors. Once the bleeding is noted, insert a 30 G needle into the posterior end of the left ventricle, and slowly inject 10 mL of pre-chilled 1x PBS to perfuse the animal.</li>
	</ol>
	</li>
	<li>Perform dorsal laminectomy15 on the mouse placed at the prone position. 
	NOTE: If cell culture is planned, spray the mouse with 70% ethanol before incision and use pre-sterilized surgical instruments for dissection.
	<ol>
		<li>Use a size 11 scalpel to make one longitudinal lateral deep incisions starting from the thoracic region down to the sacral region. Remove the skin by the scissors to expose the dorsal muscle layer.</li>
		<li>Use Friedman-Pearson Rongeur to peel off the connective tissues and muscles until the lumbosacral spine processes and bilateral transverse processes are exposed.</li>
		<li>Use a Noyes Spring Scissor to carefully open the dorsal spinal column, then switch to a Friedman-Pearson Rongeur to remove the vertebral bones to expose the spinal cord with intact spinal nerves attached.</li>
	</ol>
	</li>
	<li>Carefully dissect ipsilateral and contralateral lumbar DRG (L4/L5 DRG in our study) and place it into 1 mL of ice-cold Ca++/Mg++-free 1x HBSS in a Dounce tissue homogenizer. Now the tissues are ready for step 2. 
	NOTE: Trim the spinal nerve attached to the DRG if possible.</li>
</ol>
2. Isolate single cells from mouse lumbar DRG
<ol>
	<li>Homogenize the DRG tissue with a loose pestle in the Dounce homogenizer&#160;20-25 times.</li>
	<li>Place a sterile 70 &#956;m nylon cell strainer in a sterile 50 mL conical tube. Wet the cell strainer with 800 &#956;L of ice-cold 1x HBSS, and the flow-through is collected in the conical tube.</li>
	<li>Collect the homogenized tissue suspension from the homogenizer using a pipette and pass through the wet 70 &#956;m nylon cell strainer into the 50 mL conical tube.</li>
	<li>Rinse the homogenizer twice with 800 &#956;L of ice-cold 1x HBSS and then decant into the same 50 mL conical tube with cell strainer to increase the yield.</li>
	<li>Add 1.5 mL of equilibrated ice-cold isotonic density gradient medium (prepared in step 1.1) into a sterile 5-mL polystyrene FACS tube.</li>
	<li>Transfer the cell homogenate from the 50 mL conical tube (in step 2.4) into the FACS tube, mix well with the density gradient medium by pipetting up and down. Add an additional 500 &#956;L of 1x HBSS to seal the top.</li>
	<li>Pellet the cells by centrifugation at 800 x g for 20 min at 4 &#176;C.</li>
	<li>Carefully aspirate the supernatant containing myelin in the medium without disturbing the cell pellet at the bottom of the FACS tube.</li>
	<li>Resuspend the cells in PBS or FACS buffer containing 5% Fetal bovine serum (FBS) for FACS analysis. 
	NOTE: At least 50,000 to 100,000 cells are expected from L4 /L5 DRG of one mouse.
	<ol>
		<li>Resuspend the mechanically isolated DRG cells (L4/L5) in 100 &#956;L of PBS containing 5% Fetal Bovine Serum and then incubate with &#945;-mouse CX3CR1-APC antibody (1:2,000) in the dark at 4 &#176;C for 1 h.</li>
		<li>Wash the cells with 5 mL of PBS once; centrifuge the cells 360 x g for 8 min at 4 &#176;C.</li>
		<li>Aspirate the supernatant, then resuspend the cell pellet in 300 &#956;L of PBS for FCAS analysis. If cell sorting is planned, resuspend the cells in the FACS buffer instead.</li>
	</ol>
	</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+functional+types+of+macrophage+in+dorsal+root+ganglia+and+spinal+nerves+proximal+to+sciatic+and+spinal+nerve+transections+in+the+rat.">Distinct functional types of macrophage in dorsal root ganglia and spinal nerves proximal to sciatic and spinal nerve transections in the rat.</a> Experimental Neurology. 184 (2), 590-605 (2003).</li><li>Simeoli, R., Montague, K., Jones, H. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomal+cargo+including+microRNA+regulates+sensory+neuron+to+macrophage+communication+after+nerve+trauma.">Exosomal cargo including microRNA regulates sensory neuron to macrophage communication after nerve trauma.</a> Nature Communication. 8 (1), 1778 (2017).</li><li>Cobos, E. J., Nickerson, C. 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J., Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+macrophage+ablation+in+transgenic+mice+expressing+a+Fas-based+suicide+gene.">Conditional macrophage ablation in transgenic mice expressing a Fas-based suicide gene.</a> J Leukoc Biol. 75 (4), 612-623 (2004).</li><li>Lopes, D. M., Malek, N., Edye, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+differences+in+peripheral+not+central+immune+responses+to+pain-inducing+injury.">Sex differences in peripheral not central immune responses to pain-inducing injury.</a> Science Reports. 7 (1), 16460 (2017).</li><li>Kim, Y. 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The authors declare no competing financial interests related to this manuscript.

Here we introduce a new method to effectively enrich isolated macrophages from mouse DRG. The conventional approach to isolate DRG immune cells requires enzymatic digestion15,18, which is now replaced with mechanical homogenization in our protocol to limit undesired cell damage and increase the yield. Therefore, the new protocol is far less time consuming. More importantly, enzyme digestion might stimulate the macrophages and change the molecular signature. In co...

There is now considerable evidence that immune cells contribute to the neuropathic pain following peripheral nerve injury1,2. Peripheral monocytic cells, including mature macrophages, are known to respond to tissue injury and systemic infection through phagocytosis, antigen presentation, and cytokine release. Paralleling the nerve injury-induced microglia activation in the spinal dorsal horn, macrophages in the dorsal root ganglia (DRG) also expand significantly after nerve injury3,4. Notably, there are growing interests to determine if macrophages contribute to neuropathic pain development after peripheral nerve injury by interacting with sensory neurons in the DRG5,6,7,8,9,10,11. Moreover, recent studies also implicate the contribution of DRG macrophages in the axonal repair after nerve injury12,13. Another study further suggests that macrophage subpopulations (i.e., CD11b+Ly6Chi and CD11b+Ly6Clow/- cells) may play a different role in the mechanical hypersensitivity14. Therefore, rapidly phenotyping the response of DRG macrophages in the context of nerve injury may help us identify neuroimmune factors contributing to neuropathic pain.
Conventionally, the protocol to isolate macrophages in the DRG involves multiple steps including enzymatic digestion15,16. The technique is often time consuming and can be costly for large-scale experiments. Although mild digestion with collagenase type II (4 mg/mL) and dispase type II (4.7 mg/mL) for 20 min was recommended previously15, it is conceivable that cells after the exposure to this enzyme are prone to cell damage or cell death, which may lead to low yield. In addition, the difference in the quality of enzymes from batch to batch may further impact the efficiency of this process. More importantly, macrophages exposed to the enzyme digestion might be undesirably stimulated and thus can be very different from the in-vivo status. The changes may potentially complicate the outcome of the functional study.
Here we describe an enzyme-free protocol to rapidly isolate DRG macrophages at 4 &#176;C using mechanical dissociation. The samples are kept on ice to limit cellular stress. As a result, our approach provides an advantage to maintain consistency of the isolation, and the isolated cells are presumably healthier and less stimulated. We further present the evidence to validate the quality of the isolated cells with Fluorescence-activated Cell Sorting (FACS) analysis.

<table>
	<tbody>
		<tr>
			<td>AP20187</td>
			<td>Clontech</td>
			<td>635058</td>
			<td></td>
		</tr>
		<tr>
			<td>a-mouse CX3CR1-APC antibody</td>
			<td>Biolegend</td>
			<td>149007</td>
			<td></td>
		</tr>
		<tr>
			<td>Avertin</td>
			<td>Sigma</td>
			<td>T48402</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer (70 mm nylon)</td>
			<td>Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td></td>
		</tr>
		<tr>
			<td>Dounce tissue homogenizer</td>
			<td>Wheaton</td>
			<td>357538 (1ml)</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS tubes (5ml)</td>
			<td>Falcon</td>
			<td>352052</td>
			<td></td>
		</tr>
		<tr>
			<td>Friedman-Pearson Rongeur</td>
			<td>FST</td>
			<td>16121-14</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS (10x, Ca++/Mg++-free)</td>
			<td>Gibco</td>
			<td>14185-052</td>
			<td></td>
		</tr>
		<tr>
			<td>Noyes Spring Scissor</td>
			<td>FST</td>
			<td>15012-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>Sigma</td>
			<td>P4937-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>Sigma</td>
			<td>P4864-10ml</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

To validate the isolated cells, we first chose the Macrophage Fas-Induced Apoptosis (MAFIA) transgenic mice17. This line expresses a drug-inducible FK506-binding protein (FKBP)-Fas suicide fusion gene and green fluorescent protein (eGFP) under the control of the promoter of CSF1 receptor (CSF1R), which is specifically expressed in both macrophages and microglia. Systemic injection of FK-binding protein dimerizer, AP20187 (AP), induces the apoptosis of the cells expressing the transgene. The expres...

Watch this Scientific Journal Video about Rapid Isolation of Dorsal Root Ganglion Macrophages at JoVE.com

Rapid Isolation of Dorsal Root Ganglion Macrophages

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 ...

rapid-isolation-of-dorsal-root-ganglion-macrophages

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Neuroscience

10.1K Views.  University of California San Francisco. Here we present a mechanical dissociation protocol to rapidly isolate macrophages from the dorsal root ganglion for phenotyping and functional analysis.

Video: Rapid Isolation of Dorsal Root Ganglion Macrophages

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 ...

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These neurotropic cancer cells have a unique ability to migrate unidirectionally along nerves towards the central nervous system (CNS). The dorsal root ganglia (DRG)/cancer cell model is a three dimensional (3D) in vitro model frequently used for studying the interaction between neural stroma and cancer cells. In this model, mouse or human cancer cell lines are grown in ECM adjacent to preparations of freshly dissociated cultured DRG. In this article, the DRG isolation protocol from mice, and implantation in petri dishes for co-culturing with pancreatic cancer cells are demonstrated. Five days after implantation, the cancer cells made contact with the DRG neurites. Later, these cells formed bridgeheads to facilitate more extensive polarized, neurotropic migration of cancer cells.

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

This video article shows the use of the dorsal root ganglia (DRG)/cancer cell model in pancreatic ductal adenocarcinoma.

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These ...

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 ...

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of the PNS are affected by injury and disease, affecting the nerve function and the capacity for regeneration. Neuronal immune cells are commonly analyzed by immunofluorescence (IF). While IF is essential for determining the location of the immune cells in the nerve, IF is only semi-quantitative and the method is limited to the number of markers that can be analyzed simultaneously and the degree of surface expression. In this study, flow cytometry was used for quantitative analysis of leukocyte infiltration into sciatic nerves or dorsal root ganglions (DRGs) of individual mice. Single cell analysis was performed using DAPI and several proteins were analyzed simultaneously for either surface or intracellular expression. Both sciatic nerves from one mouse that were treated according to this protocol generated &#8805; 30,000 single nucleated events. The proportion of leukocytes in the sciatic nerves, determined by expression of CD45, was approximately 5% of total cell content in the sciatic nerve and approximately 5-10% in the DRG. Although this protocol focuses primarily on the immune cell population within the PNS, the flexibility of flow cytometry to measure a number of markers simultaneously means that the other cells populations present within the nerve, such as Schwann cells, pericytes, fibroblasts, and endothelial cells, can also be analyzed using this method. This method therefore provides a new means for studying systemic effects on the PNS, such as neurotoxicology and genetic models of neuropathy or in chronic diseases, such as diabetes.

Quantitative analysis of cell content within the murine sciatic nerve is difficult due to the scarcity of the tissue. This protocol describes a method for tissue digestion and preparation that provides sufficient cells for flow cytometry analysis of immune cell populations from nerves of individual mice.

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of ...

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.