Name: Video: Recolección de DRG de la columna vertebral de la rata: un procedimiento para extraer el ganglio de la raíz dorsal de la columna vertebral de un modelo de rata - Concepto
Uploaded: 2023-04-30T13:40:19.000+00:00
Duration: 1 min 30 s
Description: 4.7K vistas. Fuente: Schmidt, L. B. et al. El modelo in vivo de membrana corioalantoica de pollito para evaluar la invasión perineural en el cáncer de cabeza y cuello. J. Vis. Exp. (2019) En este video, demostramos la extracción del ganglio de la raíz dorsal o DRG de la columna vertebral de una rata cosechada para aplicaciones posteriores.

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Harvest and Preparation of DRGs (estimated timing: 2 h, day 8)
NOTE: Experiments with mice and rats require approval from the IACUC. In some countries, the use of chicken eggs also requires approval.
<ol>
	<li>Get six to seven week-old Sprague Dawley rats (~200 g in weight) to extract DRGs. 
	NOTE: One rat should yield ~40 cervical and thoracic DRGs. Mouse DRG also integrates in the CAM; however, the conditions for this species need to be optimized independently.</li>
	<li>In a laminar flow cabinet, harvest DRGs from cervical and thoracic regions following the protocol for mouse DRG extraction published elsewhere. Follow Figure 1 for orientation on how to harvest DRGs.
	<ol>
		<li>Euthanize the rat by cardiac puncture after administration of Ketamine/ Xylazine injected intraperitoneally. Clean the rat skin with 70% ethanol and remove the rat spine using a scissor. Do not perform cervical dislocation because this would damage the cervical DRGs.</li>
		<li>Separate the cervical, thoracic, and lumbar regions with the same scissor, following the schematic anatomic representation and gross images provided in the Figure 1A-D. Place the spine sections in a 10 cm culture dish with 1X PBS to keep tissues wet.</li>
		<li>With a delicate bone scissor, open the vertebral bones in the dorsal and ventral aspects, separating the spine in two lateral halves (Figure 1E-F).&#160; Place the tissue sections in a clean 10 cm dish with fresh 1X PBS.</li>
		<li>Using forceps, gently detach the spinal cord from the vertebral bones to visualize the DRGs (Figure 1G).</li>
		<li>With fine forceps held underneath each DRG, grasp it and pull it out from the bone cavity in which it is lodged. Do not hold the DRG directly because this will cause tissue damage. Do not trim the axon bundles from the DRG (Figure 1H). 
		NOTE: Avoid using lumbar DRGs since these have reduced integration in the CAM. For DRG region location, follow the schematic illustration and gross anatomic images in Figure 1A-D.</li>
	</ol>
	</li>
	<li>Immediately after harvesting, place each DRG into DMEM culture medium supplemented with 2% Penicillin/Streptomycin (Pen/Strep) and 10% heat-inactivated Fetal Bovine Serum (FBS) to help prevent bacterial contamination of the DRGs. Group all DRGs in the same 6 cm culture dish with 4 mL of culture medium.</li>
	<li>After harvesting all DRGs, transfer them to a new culture dish with DMEM culture medium supplemented with 2% Pen/Strep plus 10% FBS and containing 1.25 &#181;g/mL of red fluorescent dye. Incubate for 1 h in the cell culture incubator.</li>
</ol>

<table><tbody><tr><td>PBS (1x) pH 7.4</td><td>Gibco</td><td># 10010-023</td><td>Phosphate Buffered Saline</td></tr><tr><td>Sprague Dawley rats (females)</td><td>Charles River laboratories</td><td>Strain code: 001</td><td>6-7 weeks old (190-210g in weight)</td></tr><tr><td>Dumont # 5 fine forceps</td><td>Fine Science Tools (FST)</td><td># 11254-20</td><td>Used to harvest DRG</td></tr><tr><td>Extra fine Graefe forceps, curved</td><td>Fine Science Tools (FST)</td><td># 11151-10</td><td>Used to graft DRG onto the CAM on day 8 and to harvest CAM tissue on day 17</td></tr><tr><td>Extra fine Graefe forceps, straight</td><td>Fine Science Tools (FST)</td><td># 11150-10</td><td>Used to graft DRG onto the CAM on day 8 and to harvest CAM tissue on day 17</td></tr><tr><td>DMEM (1x)</td><td>Gibco</td><td># 11965-092</td><td>Dulbeecco`s Modified Eagle Medium</td></tr><tr><td>CellTracker Red CMTPX fluorescent dye</td><td>Life Technologies</td><td># C34552</td><td>Reconstitute 50&amp;micro;g in 40&amp;micro;L of DMSO and stock at -20oC. Use 1&amp;micro;L of stock solution/mL of culture medium</td></tr><tr><td>Pen/Strep</td><td>Gibco</td><td># 15140-122</td><td>10,000 Units/mL Penicilin, 10,000 &amp;micro;g/mL Streptomycin</td></tr><tr><td>HI FBS</td><td>Gibco</td><td># 10082-147</td><td>Heat-inactivated Fetal Bovine Serum</td></tr><tr><td>DMSO</td><td>Fisher Bioreagents</td><td># BP231-100</td><td>Dimethyl Sulfoxide</td></tr></tbody></table>

harvesting drg from rat spine: a procedure to extract dorsal root ganglion from the spine of a rat model

Source: Schmidt, L. B. et al. <a href="https://www.jove.com/video/59296" target="_blank">The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer.</a> J. Vis. Exp. (2019)
In this video, we demonstrate the extraction of dorsal root ganglion or DRG from a harvested rat spine for downstream applications.

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/20476/20476fig1.jpg" /> 
Figure 1: DRG extraction on day 8. A. Rat schematic illustrating the anatomical location of the spine. B. Diagram of the rat vertebrae configuration showing different body regions; green for cervical, dark blue for thoracic, orange for lumbar and light blue for sacral vertebrae. C-D. Ventral aspect of the rat spine after surgical exci...

Harvesting DRG from Rat Spine: A Procedure to Extract Dorsal Root Ganglion from the Spine of a Rat Model

Source: Schmidt, L. B. et al. The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer. J. Vis. Exp. (2019)
...

The dorsal root ganglia or DRG are clusters of cell bodies of sensory neurons lodged within the vertebrae on either side of the spinal cord. Their primary function is to transmit sensory neuronal signals from the peripheral nervous system to the central nervous system.
To isolate the dorsal root ganglia, begin by taking a freshly harvested rat spine. Dissect the spine to separate the cervical, thoracic, and lumbar regions. Keep the spine sections in a&#160; suitable buffer to ensure tissue hydration.
Now, position the thoracic section with its dorsal side facing up. Divide the section into two lateral halves. Next, secure the dissected half of the spine segment, medial side up. Gently detach the bisected spinal cord - a long, tubular structure - from the segmented vertebral bones of the section to reveal the underlying DRG.
The DRG appear as distinct white masses with dorsal and ventral roots embedded within the spinal canal. With fine forceps, carefully pull out the DRG to dislodge it from the spinal canal. Finally, transfer the harvested DRG into a suitable media for further downstream applications.

harvesting-drg-from-rat-spine-procedure-to-extract-dorsal-root

Research

JoVE Encyclopedia of Experiments

Cancer Research

Head and Neck Cancer

Assays & techniques

4.7K vistas. Fuente: Schmidt, L. B. et al. El modelo in vivo de membrana corioalantoica de pollito para evaluar la invasión perineural en el cáncer de cabeza y cuello. J. Vis. Exp. (2019) En este video, demostramos la extracción del ganglio de la raíz dorsal o DRG de la columna vertebral de una rata cosechada para aplicaciones posteriores. 

Video: Recolección de DRG de la columna vertebral de la rata: un procedimiento para extraer el ganglio de la raíz dorsal de la columna vertebral de un modelo de rata - Concepto

A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, radiation, and chemotherapy, locoregional recurrences and distant metastases occur at higher rates, and overall survival is decreased by 40% compared to HNSCC without PNI. In vitro studies of the pathways involved in HNSCC PNI have historically been challenging given the lack of a consistent, reproducible assay. Described here is the adaptation of the dorsal root ganglion (DRG) assay for the examination of PNI in HNSCC. In this model, DRG are harvested from the spinal column of a sacrificed nude mouse and placed within a semisolid matrix. Over the subsequent days, neurites are generated and grow in a radial pattern from the cell bodies of the DRG. HNSCC cell lines are then placed peripherally around the matrix and invade preferentially along the neurites toward the DRG. This method allows for rapid evaluation of multiple treatment conditions, with very high assay success rates and reproducibility.

Perineural invasion (PNI) is a common feature of head and neck squamous cell carcinoma (HNSCC), conferring lower survival rates. Its mechanisms are poorly understood. Utilizing neurites generated from murine dorsal root ganglia confined to a semisolid matrix, the pathways involved in the PNI of HNSCC cell lines can be investigated.

Un modelo para la invasión perineural en cabeza y cuello Carcinoma de células escamosas

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, ...

Investigación

JoVE Journal

Investigación sobre el cáncer

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

Cancer cells invade nerves through a process termed perineural invasion (PNI), in which cancer cells proliferate and migrate in the nerve microenvironment. This type of invasion is exhibited by a variety of cancer types, and very frequently is found in pancreatic cancer. The microscopic size of nerve fibers within mouse pancreas renders the study of PNI difficult in orthotopic murine models. Here, we describe a heterotopic in vivo model of PNI, where we inject syngeneic pancreatic cancer cell line Panc02-H7 into the murine sciatic nerve. In this model, sciatic nerves of anesthetized mice are exposed and injected with cancer cells. The cancer cells invade in the nerves proximally toward the spinal cord from the point of injection. The invaded sciatic nerves are then extracted and processed with OCT for frozen sectioning. H&amp;E and immunofluorescence staining of these sections allow quantification of both the degree of invasion and changes in protein expression. This model can be applied to a variety of studies on PNI given its versatility. Using mice with different genetic modifications and/or different types of cancer cells allows for investigation of the cellular and molecular mechanisms of PNI and for different cancer types. Furthermore, the effects of therapeutic agents on nerve invasion can be studied by applying treatment to these mice.

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

We describe an in vivo murine model of perineural invasion by injecting syngeneic pancreatic cancer cells into the sciatic nerve. The model allows for quantification of the extent of nerve invasion, and supports investigation of the cellular and molecular mechanisms of perineural invasion.

Un modelo de nervio ciático murinos In Vivo de invasión Perineural

Cancer cells invade nerves through a process termed perineural invasion (PNI), in which cancer cells proliferate and migrate in the nerve ...

Using the Chicken Chorioallantoic Membrane In Vivo Model to Study Gynecological and Urological Cancers

Mouse models are the benchmark tests for in vivo cancer studies. However, cost, time, and ethical considerations have led to calls for alternative in vivo cancer models. The chicken chorioallantoic membrane (CAM) model provides an inexpensive, rapid alternative that permits direct visualization of tumor development and is suitable for in vivo imaging. As such, we sought to develop an optimized protocol for engrafting gynecological and urological tumors into this model, which we present here. Approximately 7 days postfertilization, the air cell is moved to the vascularized side of the egg, where an opening is created in the shell. Tumors from murine and human cell lines and primary tissues can then be engrafted. These are typically seeded in a mixture of extracellular matrix and medium to avoid cellular dispersal and provide nutrient support until the cells recruit a vascular supply. Tumors may then grow for up to an additional 14 days prior to the eggs hatching. By implanting cells stably transduced with firefly luciferase, bioluminescence imaging can be used for the sensitive detection of tumor growth on the membrane and cancer cell spread throughout the embryo. This model can potentially be used to study tumorigenicity, invasion, metastasis, and therapeutic effectiveness. The chicken CAM model requires significantly less time and financial resources compared to traditional murine models. Because the eggs are immunocompromised and immune tolerant, tissues from any organism can potentially be implanted without costly transgenic animals (e.g., mice) required for implantation of human tissues. However, many of the advantages of this model could potentially also be limitations, including the short tumor generation time and immunocompromised/immune tolerant status. Additionally, although all tumor types presented here engraft in the chicken chorioallantoic membrane model, they do so with varying degrees of tumor growth.

We present the chicken chorioallantoic membrane model as an alternative, transplantable, in vivo model for the engraftment of gynecological and urological cancer cell lines and patient-derived tumors.

Uso de la membrana chorioalantoica de pollo en vivo modelo para estudiar cánceres ginecológicos y urológicos

Mouse models are the benchmark tests for in vivo cancer studies. However, cost, time, and ethical considerations have led to calls for alternative in vivo ...

Harvesting DRG from Rat Spine: A Procedure to Extract Dorsal Root Ganglion from the Spine of a Rat Model

Transcripción

Harvesting Dorsal Root Ganglion from Rat Spine