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 to the ganglion roots due to shear forces. From here on, perform all steps under sterile conditions.</li>
	<li>Soak animal down with 70% ethanol. This step is important for sterilization and for preventing the hair from dragging through the inner organs.</li>
	<li>Leave the mouse to dry from ethanol. Prevent any contact of ethanol with the inner organs due to neurotoxicity.</li>
	<li>After drying the mouse, position it using pins in prone position (face down). Place the pins on each hindlimb and forelimb.</li>
	<li>Using forceps, make a midline incision extending craniocaudally from the posterior neck to the lumbar spine. Next, raise dermal flaps bilaterally using forceps and expose subcutaneous tissue.</li>
	<li>Palpate mouse skull until reaching the craniocervical junction. Using scissors perpendicular to the animal, cut through the cervical muscles and spine at the craniocervical junction (the 7th cervical vertebrae), using heavy forceps. Dissect the spine caudally and excise the spine with heavy forceps until reaching the lumbar level (hind limbs level).</li>
	<li>Perform a complete caudal transection at the lumbar spine level (the 5th lumbar vertebrae) using heavy forceps.</li>
	<li>Roll the mouse over (face up). There is no need to cover the incision since the DRG is extracted from lower vertebral levels and not the cervical.</li>
	<li>Cut down at the midline from the neck to the abdomen, retract the skin laterally and then cut to open the peritoneum.</li>
	<li>Remove the inner organs inside the peritoneum (liver, spleen, pancreas, stomach, and intestine) en bloc. Next, remove the retroperitoneal organs (i.e., kidneys and pancreas). Cranially, open the chest wall.</li>
	<li>Using forceps and a surgical blade, cut the ribs, leaving 5 mm of ribs bilaterally away from the vertebral column. At this point, the vertebral column is separated from the rest of the body.</li>
	<li>Wash the vertebral column from blood with cold (4 &#176;C) fresh phosphate buffered saline (PBS) twice.</li>
</ol>
2. Isolating the Dorsal Root Ganglia (DRG)
<ol>
	<li>Use a stereomicroscope with 4X magnification.</li>
	<li>Position the spine on a non-adherent non absorbing platform (Telfa/nylon). Position the spine face up&#160;at the same cranio-caudal orientation.</li>
	<li>Remove any spinal muscles and connective tissue. Use the ribs as a landmark for nerves as they leave the spine. Ribs also protect the nerves from the surgical tools. The DRG are located at the cervical, thoracic, and lumbar sites of the vertebrae.</li>
	<li>Next, using scissors cut the vertebral body in the midline and expose the spinal cord and DRG roots by gentle lateral retraction of the vertebral body.</li>
	<li>In the intercostal space, follow the peripheral nerve medially along the rib lateral to the DRG.</li>
	<li>Identify the DRG. It looks like the yolk of &#39;fried egg&#39; lying on the nerve.</li>
	<li>Cut the intercostal nerve distal to the DRG leaving 2-3 mm of nerve distal to the ganglia, to be used for retraction. This &#34;tail&#34; will be removed after harvesting.</li>
	<li>Grasp the nerve using forceps. Pinching the DRG body itself will cause neural cell damage and should be avoided.</li>
	<li>Approach the DRG proximally along its attachment to the spinal cord, via its anterior and posterior roots.</li>
	<li>Apply gentle retraction to the DRG by pulling the efferent nerve (intercostal nerve stump) laterally, and cut the anterior and posterior roots close to the DRG.</li>
	<li>Keep the DRG in a 35 mm Petri dish filled with fresh, ice cold DMEM supplemented with 10% fetal calf serum (FCS), 1% penicillin-streptomycin, 1% non-essential amino acids, and 1% Sodium pyruvate.</li>
</ol>
3. Implantation in the Petri Dish
<ol>
	<li>Perform the next steps on ice using pre-cooled pipette tips to prevent ECM solidification.</li>
	<li>Under a stereomicroscope at 2-4X magnification, place a 35 mm glass bottom petri dish on a paper grid. 
	IMPORTANT: Work on ice in order to keep the ECM in liquid state. Create a 1 mm diameter spot (approximately 20 &#181;l) of growth factor-depleted ECM at the center of the grid.</li>
	<li>Under direct visualization, place the DRG at the center of the ECM spot close to the bottom of the dish. 
	Note: The cancer cells used in this protocol are murine pancreatic cancer cells (KPC) established from freshly isolated tumor specimens from KPC mice as described elsewhere19.</li>
	<li>Harvest 40,000 cancer cells from confluent cultures, wash them once with PBS, and resuspend them in 40 &#181;l ECM on ice.</li>
	<li>Under direct visualization of the grid using a stereomicroscope measure 500 &#181;m at each direction from the DRG. At this point slowly seed 10,000 cells/10 &#181;l ECM. Use a 2-10 &#181;l precooled tip to inject the cells close to the dish bottom, avoiding their spread in the matrix or detachment of the matrix. (Figure 1).</li>
	<li>Leave the dish within a laminar flow hood for 10-15 min&#160;to solidify. Avoid ECM drying.</li>
	<li>Slowly add DMEM, prepared as previously mentioned, against the side wall of the plate. Add enough medium to cover the ECM (about 2 ml).</li>
	<li>Put the dish back to the incubator at 37 &#176;C.</li>
	<li>Replace the medium with fresh medium on the following day.</li>
	<li>Replace the medium every 2 days.</li>
</ol>
4. Data Acquisition, Time-lapse Videomicroscopy
<ol>
	<li>On day 7 after the implantation, take the dish for time lapse microscopy. Note that certain cells might require acquisition earlier due to faster migration.</li>
	<li>During the live imaging, place the dish in a closed environment at a mean temperature of 37 &#177;0.1 &#176;C and 5% CO2.</li>
	<li>Record the cells by using a charge coupled device camera placed on the microscope.</li>
	<li>Take digitized images every 10 min&#160;for up to 72 hr&#160;to follow cell locomotion from three to six different cells.</li>
	<li>Replace medium after 24 hr.</li>
	<li>Perform data acquisition and simple analysis using the microscope software.</li>
</ol>
5. Data Analysis
<ol>
	<li>For more advanced applications (i.e., cell tracking in 2D or 3D) use specialized software (such as Imaris 4D). Note: This software determines the xy coordinates of a cell at any given time, along with the distance from origin, and speed of motion.</li>
	<li>Calculate the mean velocity by measuring the travel distance between subsequent positions in 10 min&#160;intervals. Calculate the Forward Migration Index (FMI) which is the distance of the cell front from the neurite and describes the unidirectional movement of the cells toward the DRG.</li>
</ol>

<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 research was supported by the ICRF Barbara S. Goodman endowed research career development award (2011-601-BGPC) and a grant from the US-Israel Binational Science Foundation. No other financial disclosures from the authors are to be made.

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 perineural niche microenvironment in vitro.
The protocol presented in the video has two critical steps. First, care should be taken when grasping the nerve adjacent to the DRG body (step 2.7 in the protocol). Grasping or pinching the DRG might cause irreversible mechanical or ischemic damage to the DRG preventing it from growing and sprouting when seeded in the Petri dish. The second critical step is the implantation of the cancer cells in the Petri dish inside the ECM. It is important to seed the cells slowly and with caution, to avoid the floating of cells and their spread over the Petri dish. The aim of the implantation is to localize the cells in one place, to facilitate tracking their movement (distance and direction).
Once established the model microenvironment can be modified according to the tested hypothesis. For instance, adding a third cell culture to the Petri dish (for example, non-cancerous cells) enables the researcher to compare the neurotropic migration capabilities of different cells. Furthermore, the researcher can apply different conditions (i.e., temperature, humidity, soluble factors, etc.) and examine their effect on the cells invasion ability.
The DRG model enables the researcher to study the interaction between cancer cells and nerves. It is also used for time lapse experiments in which morphological changes in the perineural niche are demonstrated in a temporal fashion. Furthermore, the neuroinvasive cells can be subjected to various treatments and their effect on the interaction with neurites of the ganglia can be assessed.
Not every cell line is suitable for the DRG model. They should be cancer cells with the intrinsic ability to invade through nerve and to degrade ECM. Furthermore, note that each cancer cell type has different invasion characteristics and so the time the cells are expected to make contact with the DRG neurites varies. For example, The KPC cells we used need about 7 days to invade through the ECM toward the DRG whereas MiaPaca cells (human pancreatic adenocarcinoma cells) need only 72-96 hr&#160;to make that contact.
A limitation of this model includes the possible incompatibility with human cells. Due to its use of mouse DRG, the mouse-human protein-protein interaction cannot always be demonstrated when human cancer cells are used. Whenever planning to use this model with the two different species, the homology of the examined protein should be tested and if there is high homology the assay is supposed to be suitable for evaluating protein-protein interactions. As well, it should kept in mind that the DRG assay as proposed in this protocol represents the neural microenvironment including other cells, not only neurons (i.e., Schwann cells, fibroblasts, macrophages, etc.). Hence, specific cell populations cannot be tested specifically.

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>
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		<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 (QLL2, B16F)&#160;(Figure 3a). Figure 3b presents a representative X and Y coordinate graph, depicting the migration path of a KPC cancer cell in contact with the nerve; time-lapse videomicroscopy analysis showed differences in the forward movement but not velocity between the KPC cells and non-invading cells (Figure 3c-d).
<img alt="Figure 1" src="/files/ftp_upload/52990/52990fig1.jpg" /> 
Figure 1: Schematic Illustration of the Protocol Steps. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52990/52990fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52990/52990fig2.jpg" /> 
Figure 2: Cancer Cell Invasion Along the Neurons of the DRG. (a) DRG (top) and cancer cells (bottom) on day 0 after seeding&#160;(5X magnification).&#160; (b) DRG and cancer cells on day 7 after seeding&#160;(5X magnification). (c) Cancer cells migrate along the DRG neuron (arrows). All bars represent 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52990/52990fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52990/52990fig3.jpg" /> 
Figure 3: Dorsal Root Ganglion (DRG) Neurons Induce CNI. (a) Nerve invasion index of MiaPaCa2 cancer cells , KPC cells, QLL2, and NIH3T3 cells. (b) A coordinate graph depicting the migration path of a KPC cancer cell in contact with the nerve (red) and QLL2 cell (purple). The Y and X coordinates are shown. (n = 12-20 in each group). (c) Analysis of distance from origin of migrating QLL2 cancer cells with axonal contact and (d) KPC cancer cells (n = 20). The direction of migration was constantly toward the nerve ganglion. P values in (a) were calculated by two-sided Student t test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52990/52990fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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9.0K Views.  Rambam Medical Healthcare Campus. This video article shows the use of the dorsal root ganglia (DRG)/cancer cell model in pancreatic ductal adenocarcinoma.

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

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 ...

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 ...

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of primary afferent fibers mediated by GABAergic axo-axonal synapses (primary afferent depolarization). The strength of primary afferent depolarization can be measured by recording of volume-conducted potentials at the dorsal root (dorsal root potentials, DRP). Pathological changes of presynaptic inhibition are crucial in the abnormal central processing of certain pain conditions and in some disorders of motor hyperexcitability. Here, we describe a method of recording DRP in vivo in mice. The preparation of spinal cord dorsal roots in the anesthetized animal and the recording procedure using suction electrodes are explained. This method allows measuring GABAergic DRP and thereby estimating spinal presynaptic inhibition in the living mouse. In combination with transgenic mouse models, DRP recording may serve as a powerful tool to investigate disease-associated spinal pathophysiology. In vivo recording has several advantages compared to ex vivo isolated spinal cord preparations, e.g. the possibility of simultaneous recording or manipulation of supraspinal networks and induction of DRP by stimulation of peripheral nerves.

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

GABAergic presynaptic inhibition is a powerful inhibitory mechanism in the spinal cord important for motor and sensory signal integration in spinal cord networks. Underlying primary afferent depolarization can be measured by recording of dorsal root potentials (DRP). Here we demonstrate a method of in vivo recording of DRP in mice.

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of ...

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 ...

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 ...