Name: in vitro modeling of cancerous neural invasion: the dorsal root ganglion model
Uploaded: 2016-04-12T14:00:00
Description: Watch this Scientific Journal Video about In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model at JoVE.com

Edith Suss-Toby is thanked for her assistance in the time-lapse microscopy and image analysis. Nofar Rada is thanked for the artistic work.

Four- to six-week-old female C57BL/CJ mice (Harlan, Jerusalem, Israel) were used in the experiment according to the Association for Assessment and Accreditation of Laboratory Animal Care specifications. All experimental procedures were done in accordance with Institutional Animal Care and Use Committee and the Department of Agriculture regulations.
1. Harvesting the Spinal Cord
<ol>
	<li>Euthanize the mouse using a CO2 chamber. Avoid cervical dislocation as it might cause damage t...

<ol>
	<li>Carter, R. L., Foster, C. S., Dinsdale, E. A., Pittam, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perineural+spread+by+squamous+carcinomas+of+the+head+and+neck,+a+morphological+study+using+antiaxonal+and+antimyelin+monoclonal+antibodies.">Perineural spread by squamous carcinomas of the head and neck, a morphological study using antiaxonal and antimyelin monoclonal antibodies.</a> J Clin Pathol. 36, 269-275 (1983).</li><li>Beard, C. 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=Perineural+invasion+is+associated+with+increased+relapse+after+external+beam+radiotherapy+for+men+with+low-risk+prostate+cancer+and+may+be+a+marker+for+occult,+high-grade+cancer.">Perineural invasion is associated with increased relapse after external beam radiotherapy for men with low-risk prostate cancer and may be a marker for occult, high-grade cancer.</a> Int J Radiat Oncol Biol Phys. 58, 19-24 (2004).</li><li>Maru, N., Ohori, M., Kattan, M. W., Scardino, P. T., Wheeler, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognostic+significance+of+the+diameter+of+perineural+invasion+in+radical+prostatectomy+specimens.">Prognostic significance of the diameter of perineural invasion in radical prostatectomy specimens.</a> Hum Pathol. 32, 828-833 (2001).</li><li>Ceyhan, G. O., 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=Pancreatic+neuropathy+and+neuropathic+pain--a+comprehensive+pathomorphological+study+of+546+cases.">Pancreatic neuropathy and neuropathic pain--a comprehensive pathomorphological study of 546 cases.</a> Gastroenterology. 136, 177-186 (2009).</li><li>Ceyhan, G. O., 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=The+neurotrophic+factor+artemin+promotes+pancreatic+cancer+invasion.">The neurotrophic factor artemin promotes pancreatic cancer invasion.</a> Ann Surg. 244, 274-281 (2006).</li><li>Takahashi, T., 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=Perineural+invasion+by+ductal+adenocarcinoma+of+the+pancreas.">Perineural invasion by ductal adenocarcinoma of the pancreas.</a> J Surg Oncol. 65, 164-170 (1997).</li><li>Zhu, Z., 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=Nerve+growth+factor+expression+correlates+with+perineural+invasion+and+pain+in+human+pancreatic+cancer.">Nerve growth factor expression correlates with perineural invasion and pain in human pancreatic cancer.</a> J Clin Oncol. 17, 2419-2428 (1999).</li><li>Hirai, I., 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=Perineural+invasion+in+pancreatic+cancer.">Perineural invasion in pancreatic cancer.</a> Pancreas. 24, 15-25 (2005).</li><li>Mitchem, J. B., 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=Targeting+tumor-infiltrating+macrophages+decreases+tumor-initiating+cells,+relieves+immunosuppression,+and+improves+chemotherapeutic+responses.">Targeting tumor-infiltrating macrophages decreases tumor-initiating cells, relieves immunosuppression, and improves chemotherapeutic responses.</a> Cancer Res. 73, 1128-1141 (2013).</li><li>Kelly, K., 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=Attenuated+multimutated+herpes+simplex+virus-1+effectively+treats+prostate+carcinomas+with+neural+invasion+while+preserving+nerve+function.">Attenuated multimutated herpes simplex virus-1 effectively treats prostate carcinomas with neural invasion while preserving nerve function.</a> FASEB J. 22, 1839-1848 (2008).</li><li>Dai, H., 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=Enhanced+survival+in+perineural+invasion+of+pancreatic+cancer,+an+in+vitro+approach.">Enhanced survival in perineural invasion of pancreatic cancer, an in vitro approach.</a> Hum Pathol. 38, 299-307 (2007).</li><li>Ayala, G. E., 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=Cancer-related+axonogenesis+and+neurogenesis+in+prostate+cancer.">Cancer-related axonogenesis and neurogenesis in prostate cancer.</a> Clin Cancer Res. 14, 7593-7603 (2008).</li><li>Ayala, G. E., 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=Stromal+antiapoptotic+paracrine+loop+in+perineural+invasion+of+prostatic+carcinoma.">Stromal antiapoptotic paracrine loop in perineural invasion of prostatic carcinoma.</a> Cancer Res. 66, 5159-5164 (2006).</li><li>Ceyhan, G. O., 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=Neural+invasion+in+pancreatic+cancer,+a+mutual+tropism+between+neurons+and+cancer+cells.">Neural invasion in pancreatic cancer, a mutual tropism between neurons and cancer cells.</a> Biochem Biophys Res Commun. 374, 442-447 (2008).</li><li>Bapat, A. A., Hostetter, G., Von Hoff, D. D., Han, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perineural+invasion+and+associated+pain+in+pancreatic+cancer.">Perineural invasion and associated pain in pancreatic cancer.</a> Nat Rev Cancer. 11, 695-707 (2011).</li><li>Ketterer, K., 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=Reverse+transcription-PCR+analysis+of+laser-captured+cells+points+to+potential+paracrine+and+autocrine+actions+of+neurotrophins+in+pancreatic+cancer.">Reverse transcription-PCR analysis of laser-captured cells points to potential paracrine and autocrine actions of neurotrophins in pancreatic cancer.</a> Clin Cancer Res. 9, 5127-5136 (2003).</li><li>Gil, Z., 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=Nerve-sparing+therapy+with+oncolytic+herpes+virus+for+cancers+with+neural+invasion.">Nerve-sparing therapy with oncolytic herpes virus for cancers with neural invasion.</a> Clin Cancer Res. 13, 6479-6485 (2007).</li><li>Gil, Z., 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=Paracrine+regulation+of+pancreatic+cancer+cell+invasion+by+peripheral+nerves.">Paracrine regulation of pancreatic cancer cell invasion by peripheral nerves.</a> J Natl Cancer Inst. 102, 107-118 (2010).</li><li>Weizman, N., 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=Macrophages+mediate+gemcitabine+resistance+of+pancreatic+adenocarcinoma+by+upregulating+cytidinedeaminase.">Macrophages mediate gemcitabine resistance of pancreatic adenocarcinoma by upregulating cytidinedeaminase.</a> Oncogene. 33, 3812-3819 (2014).</li></ol>

This article presents an in vitro model that recapitulates the cancerous microenvironment in the neural niche, the DRG model. The video demonstrates all the steps starting from recognizing anatomical landmarks such as the DRG in the mouse, its extraction, and finally, its culturing in ECM. Co-culturing the DRG alongside with cancer cells is also presented. There are no other models for in vitro perineural invasion research described in the literature making this model essential for studying the perineur...

Solid tumors disseminate in three main ways: direct invasion, lymphatic spread, and hematogenic spread. However, there is a fourth means of cancer spread that is frequently disregarded, dissemination along nerves. Cancerous neural invasion (CNI) is a well-known route of cancer spread, especially in cancers of the head and neck,1 prostate2, 3 and pancreas.4-8 CNI occurs in more than 80% of individuals with pancreatic adenocarcinoma, leading to retroperitoneal tumor spread through celiac ganglion nerves. These neurotropic cancer cells have a unique ability to migrate unidirectionally along nerves towards the central nervous system (CNS).9 This finding suggests that the perineural microenvironment can be exploited by cancer cells, providing factors that support malignant growth.
One of the few in vitro models for CNI research is the dorsal root ganglia (DRG)/cancer cell model. This model is frequently used for studying the paracrine interaction between neural stroma and cancer cells.10-18 In this model, mouse or human cancer cell lines are grown in extracellular matrix (ECM) adjacent to preparations of freshly dissociated cultured DRG.
This video article shows the application of in vitro CNI in pancreatic ductal adenocarcinoma.

<table border="0" cellpadding="0" cellspacing="0" width="710">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:417px;">Equipments:</td>
			<td style="width:121px;"></td>
			<td style="width:108px;"></td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Operating microscope</td>
			<td>Leica</td>
			<td>M205</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tiime Lapse System</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Forceps</td>
			<td>Sigma-Aldrich</td>
			<td>F4142&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical blade</td>
			<td>Sigma-Aldrich</td>
			<td>Z309036</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scissors</td>
			<td>Sigma-Aldrich</td>
			<td>S3271</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">35mm petri dishes, glass bottom</td>
			<td>de groot</td>
			<td>60-627860</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company&#160;</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Materials:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">70% ethanol</td>
			<td>sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cold PBS</td>
			<td>Biological industries</td>
			<td>02-023-1A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM</td>
			<td>Biological industries</td>
			<td>01-055-1A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FCS</td>
			<td>Rhenium</td>
			<td align="right">10108165</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin and streptomycin</td>
			<td>Biological industries</td>
			<td>01-031-1B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Pyruvate</td>
			<td>Biological industries</td>
			<td>03-042-1B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-Glutamine</td>
			<td>Biological industries</td>
			<td>03-020-1B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Growth factor depleted matrigel</td>
			<td>Trevigen</td>
			<td>3433-005-01</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Using video microscopy imaging, the DRG can be seen sprouting neurites 5-7 days after implantation while the cancer cells migrating away from their colonies toward the DRG. By the 7th day after the implantation, the cancer cells come in contact with the neurites (Figure 2).
The Forward migration index of the pancreatic cancer cells used in the protocol is 3-4 fold higher than that of other cell lines ...

Watch this Scientific Journal Video about In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model at JoVE.com

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.

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 ...

The overall goal of this model is to recapitulate the cancerous neural microenvironment. This enables exploration of cancer cell neuron interactions. This method can help answer key questions in the tumor microenvironment field, such as interaction between tumor cell and neurons and the mutual influence of each set.The main advantage of this technique is that it is reliable, easy to perform and address both cellular and cerebral factors in the neural niche. The implications of this technique extend towards therapeutic development and pharmacological targeting of key factors in the neural remodeling and invasion process throughout cancer development. After euthanizing the animal in a CO2 chamber, soak the fur with 70%ethanol and allow it to dry.Pin the mouse in a prone position, and then make a midline incision extending craniocaudally from the posterior neck to the lumber spine. Next, use forceps to raise the dermal flaps bilaterally and expose the subcutaneous tissue. Palpate the mouse skull until the craniocervical junction is identified.Use scissors angled perpendicular to the animal to cut through the cervical muscles and spine at the craniocervical junction. Then, dissect the spine caudally and use scissors to perform a complete caudal transection at the fifth lumbar vertebrae. Next, position the mouse in a supine position, and after making an incision along the midline from the neck to the abdomen, retract the skin laterally and then cut open the peritoneum.Cranially, open the chest wall with scissors. After removing the peritoneal and retroperitoneal organs, use forceps and a surgical blade to cut the ribs, leaving approximately five millimeters of ribs protruding from the vertebral column. Remove the vertebral column from the rest of the body.and wash twice with cold PBS. Position the spine facing up in the same craniocaudal orientation on a nonadherent absorbing platform under a stereo microscope equipped with 4X magnification. While looking through this stereo microscope, remove any spinal muscles and connective tissue.Use the ribs as landmarks for nerves, as they leave the spine. The DRG are located at the cervical, thoracic, and lumbar vertebrae. Use scissors to cut the vertebral body at the midline, creating a window to the spinal cord, and then gently retract the vertebral body to expose the spinal cord and the DRG roots.Next, using spring scissors, remove the upper rib to expose the DRG. Follow the peripheral nerve medially along the rib lateral to the DRG. Identify the DRG, which can be described as looking like the yolk of a fried egg lying on the nerve.Cut the intercostal nerve distal to the DRG leaving two to three millimeters of nerve distal to the ganglia to be used for retraction. Carefully grasp the efferent nerve using forceps without pinching or damaging the DRG. Now apply gentle retraction to the DRG by pulling the nerve laterally.Then cut the anterior and posterior roots close to the DRG. Transfer the isolated DRG to a 35-millimeter petri dish filled with ice-cold supplemented DMEM. Working in a laminar flow hood, place a 35-millimeter glass bottom petri dish on a paper grid on ice.Using a precooled pipette tip, dispense approximately 10 microliters of growth-factor-depleted ECM at the center of the grid. While viewing the dish through a stereo microscope under 2 to 4X magnification, place the DRG at the center of the ECM close to the bottom of the dish. After harvesting and washing 40, 000 cancer cells of interest from confluent cultures resuspend the pellet and 40 microliters of ECM on ice.Once again, view the grid through the stereo microscope, measure 500 microns from the DRG, and dispense 10 microliters of the cell suspension at this point. Repeat in all directions from the DRG. Leave the dish in the laminar flow hood for 10 to 15 minutes to solidify but not dry out.Once the ECM has solidified, slowly add supplemented DMEM by pipetting against the sidewall of the plate. Add approximately two milliliters of media, enough to cover the ECM. The addition of DMEM should be done very slowly by pipetting against the sidewall of the plate to avoid detachment of the DRG from the plate bottom.Set the plate in the tissue culture incubator, and follow the instructions in the written portion of the protocol to maintain the culture. This micrograph shows the DRG at the top of the field of view and cancer cells at the bottom of the field of view on day zero after seeding. Here, the same culture is shown seven days after seeding.As indicated by the arrows, cancer cells migrate along the DRG neuron. This histogram shows the DRG nerve invasion index of different types of cancer cells. It can be seen that Kpc 989 and MiaPaCa cells have a higher nerve invasion index than QLL2 or NIH3T3 cells.This coordinate graph depicts the migration path of a Kpc cancer cell in contact with the nerve shown in red and a QLL2 cell shown in purple. The X and Y coordinates are shown. This figure shows an analysis from distance of origin for migrating QLL2 cancer cells with axonal contact.Here the distance of origin is shown for Kpc cancer cells. In each case, the direction of migration was towards the nerve ganglion. While attempting this procedure, it is important to remember to prepare more ganglia than required since the check rate is not 100%After it's development, the DRG motive paved the way for researchers in the field of cancer research to explore the neural niche within the tumor microenvironment.After watching this video, you should have a good understanding of how to recognize anatomical landmarks such as the DRG in the mouse, extract the DRG, and finally, how to culture it in DCM. Coculturing of the DRG alongside with cancer cells is also presented.

in-vitro-modeling-cancerous-neural-invasion-dorsal-root-ganglion

Research

JoVE Journal

Neuroscience

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression (SD), as a means to develop novel therapeutic targets for episodic and chronic migraine. SD is the likely cause of migraine aura and migraine pain. It is a paroxysmal loss of neuronal function triggered by initially increased neuronal activity, which slowly propagates within susceptible brain regions. Normal brain function is exquisitely sensitive to, and relies on, coincident low-level immune signaling. Thus, neural immune signaling likely affects electrical activity of SD, and therefore migraine. Pain perception studies of SD in whole animals are fraught with difficulties, but whole animals are well suited to examine systems biology aspects of migraine since SD activates trigeminal nociceptive pathways. However, whole animal studies alone cannot be used to decipher the cellular and neural circuit mechanisms of SD. Instead, in vitro preparations where environmental conditions can be controlled are necessary. Here, it is important to recognize limitations of acute slices and distinct advantages of hippocampal slice cultures. Acute brain slices cannot reveal subtle changes in immune signaling since preparing the slices alone triggers: pro-inflammatory changes that last days, epileptiform behavior due to high levels of oxygen tension needed to vitalize the slices, and irreversible cell injury at anoxic slice centers. 
In contrast, we examine immune signaling in mature hippocampal slice cultures since the cultures closely parallel their in vivo counterpart with mature trisynaptic function; show quiescent astrocytes, microglia, and cytokine levels; and SD is easily induced in an unanesthetized preparation. Furthermore, the slices are long-lived and SD can be induced on consecutive days without injury, making this preparation the sole means to-date capable of modeling the neuroimmune consequences of chronic SD, and thus perhaps chronic migraine. We use electrophysiological techniques and non-invasive imaging to measure neuronal cell and circuit functions coincident with SD. Neural immune gene expression variables are measured with qPCR screening, qPCR arrays, and, importantly, use of cDNA preamplification for detection of ultra-low level targets such as interferon-gamma using whole, regional, or specific cell enhanced (via laser dissection microscopy) sampling. Cytokine cascade signaling is further assessed with multiplexed phosphoprotein related targets with gene expression and phosphoprotein changes confirmed via cell-specific immunostaining. Pharmacological and siRNA strategies are used to mimic and modulate SD immune signaling.

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

Migraine and its transformation to chronic migraine are immense healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression in hippocampal slice cultures, as a means to develop novel therapeutic targets.

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options.  We seek to define how neural immune ...

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 ...

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 ...

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 ...

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the dorsal root ganglion (DRGs) extends a single axon with two branches. One branch goes to the peripheral nerve (peripheral branch), and the other branch enters the spinal cord through the dorsal root (central branch). After the neural injury, the peripheral branch regenerates robustly whereas the central branch does not regenerate. Due to the high regenerative capacity, sensory axon regeneration has been widely used as a model system to study mammalian axon regeneration in both the peripheral nervous system (PNS) and the central nervous system (CNS). Here, we describe a previously established approach protocol to manipulate gene expression in mature sensory neurons in vivo via electroporation. Based on transfection with plasmids or small RNA oligos (siRNAs or microRNAs), the approach allows for both loss- and gain-of-function experiments to study the roles of genes-of-interests or microRNAs in regulation of axon regeneration in vivo. In addition, the manipulation of gene expression in vivo can be controlled both spatially and temporally within a relatively short time course. This model system provides a unique tool to investigate the molecular mechanisms by which mammalian axon regeneration is regulated in vivo.

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an effective approach to deliver genes of interests into cells. By applying this approach in vivo on the neurons of adult mouse dorsal root ganglion (DRG), we describe a model to study axon regeneration in vivo.

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...

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 ...

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 ...