Name: an in vivo murine sciatic nerve model of perineural invasion
Uploaded: 2018-04-23T13:00:00.000+00:00
Duration: 9 min 55 s
Description: Watch this Scientific Journal Video about An In Vivo Murine Sciatic Nerve Model of Perineural Invasion at JoVE.com

The authors acknowledge the technical services provided by the molecular cytology facility and the animal facility of Memorial Sloan Kettering Cancer Center. This work was supported by NIH grants CA157686 (to R.J. Wong) and P30 CA008748 (Memorial Sloan Kettering Cancer Center support grant).

All of the procedures with animal subjects were approved by the Institutional Animal Care and Use Committee at Memorial Sloan Kettering Cancer Center.
1. Preparation of the Cancer Cells
<ol>
	<li>Harvest sub-confluent Panc02-H7 cells with 0.25% trypsin for 5 min at 37 &#176;C. Collect the cells in a 15 mL centrifuge tube. 
	NOTE: The cells are grown in T-225 flask, which contains about 12 x 106 cells per flask at 80% confluency and 4 mL trypsin/ flask is used.</li>
	<li>Centrifuge the cells at x 900 g at 4&#176;C for 5 min. Wash the cells by resuspending the cell pellet in 1 mL of PBS (by pipetting up and down twice), and transfer the suspension to a 1.5 mL microcentrifuge tube on ice.</li>
	<li>Centrifuge the cells again at x 900 g at 4&#176;C for 5 min, and discard the supernatant without disturbing the pellet. Keep the pelleted cells on ice until injection.</li>
</ol>
2. Preparation of the Mice and Surgery
Note: 8-week-old, male and female C57BL/6J mice are used in this study. The surgery conditions follows the IACUC rules of our institution. The instruments are sterilized, the surgical working surface is disinfected, the animal is disinfected and the surgeon wears sterile gloves.
<ol>
	<li>On the day before the surgery, anesthetize the mouse using 2% isoflurane and then&#160;remove the fur along the length of femur on the dorsal side with either a thin razor or a chemical hair removal agent.</li>
	<li>On the day of the surgery, anesthetize the mice using 2% isofluorane in an induction chamber. Confirm the anesthetization by a toe pinch stimulus and a lack of response.</li>
	<li>Apply vet ointment on eyes to prevent dryness under anesthesia.</li>
	<li>Place the anesthetized mouse on its ventral side, and gently secure each limb with hypoallergenic tape to create mild tension in the limb to be injected.&#160;Anesthesia is maintained using isoflurane delivered via a precision vaporizer and nose cone.</li>
	<li>Clean the injection site with Betadine, then again with 70% alcohol. Repeat this process two more times. Make sure that no loose hair remains on the surgical field.</li>
	<li>Make a 1 cm incision with small scissors about 2 mm below and parallel to the femur. Retract the skin with forceps laterally to expose the muscles underneath.</li>
	<li>The sciatic nerve runs deep to the gluteus maximus and biceps femoris muscles. Separate these two muscles along a fascial plane with small scissors and expose the sciatic nerve underneath. Free the nerve from the surrounding muscles using blunt dissection.</li>
	<li>Draw 3 &#956;L of cancer cells (about 50,000 cells) from the pellet into a 10 &#956;L syringe. 
	NOTE: Alternatively, draw 3 &#956;L of PBS as a control.</li>
	<li>Place a small metal spatula underneath the nerve at the point of injection for support. Under visualization with a dissecting microscope, insert the needle into the nerve against the metal base, keeping the needle as parallel to the nerve as possible upon insertion. Be careful as not to puncture through the back of the nerve. Minimize the handling of nerve as much as possible throughout this process.</li>
	<li>Slowly inject into the nerve over 5 s. A formation of a bulb in the injection area indicates a good injection. Then leave the needle in place for 3 s before removing the needle gently. Keeping the needle in place for 3 s minimizes backflow of the cells out of the nerve. 
	NOTE: Injection can be performed toward the distal nerve or the spinal cord. It is important to remain consistent within a set of experiments. If the cells spill outside, the animal should not be included in the analysis of the experiment. With experience, these events are very rare.</li>
	<li>Return the nerve to its original position. Cover the nerve with the overlying muscles. Treat the mice with proper analgesia, and then close the skin with 5-0 Nylon sutures.</li>
	<li>Place the mouse alone in a clean cage for observation during recovery, until it fully awakens from anesthesia. 
	NOTE: It takes 5-15 min for the animal to regain full consciousness. The animal is not left unattended until it has regained sufficient consciousness to maintain sternal recumbency. The animal is not returned to the company of other animals until fully recovered. Thereafter, evaluate recovery at least once every 24 h for 72 h.</li>
</ol>
3. Sciatic Nerve Extraction
<ol>
	<li>On postop day #7, euthanize the mice with CO2. Place the mouse on its ventral side, and stabilize the distal limbs using pins.</li>
	<li>Remove the skin on the dorsal side of the injected limb and torso.</li>
	<li>Using blunt dissection, expose the sciatic nerve deep to the muscles.</li>
	<li>The nerve courses between the ilium and the sacrum. To have access to the nerve at the spinal cord region, separate the two bones. First insert closed scissors in the narrow area where the sciatic nerve is located and then open the scissors while holding the mouse. Be delicate and maintain the integrity of the sciatic nerve during the dissection.</li>
	<li>Carefully dissect the sciatic nerve distally to the end of the femur, and proximally to the spinal cord.</li>
	<li>NOTE: Invaded nerves are extremely fragile and prone to breaking under tension or forceful handling.</li>
	<li>Harvest the nerve by first cutting its distal end. Carefully lift the nerve while freeing it from adjacent tissue. Cut the nerve at the proximal end, as close as possible to its exit from the spinal column.</li>
	<li>Record then gross length of invasion using a Vernier caliper. This macroscopic estimation is only indicative.</li>
</ol>
4. Nerve Processing and Quantification
<ol>
	<li>Embed the dissected nerve in OCT compound. Make sure to place nerves longitudinally and as flat on the bottom of the mold as possible.</li>
	<li>Indicate on the cassette the proximal and distal side of the nerve by marking the letter P and D.</li>
	<li>Place embedded nerves on top of dry ice, if they are not sectioned immediately. Sample could be preserved at -80 &#176;C for several weeks.</li>
	<li>Section samples using a Cryostat microtome at 5 &#956;m thickness and place sections on glass slides. If possible, fit 2 nerve sections per slide. Indicate proximal side of the nerve.</li>
	<li>Stain slides using H&#38;E staining12.</li>
	<li>Digitally scan stained nerve sections with a slide-scanner that provides high-resolution digital data.</li>
	<li>Using imaging software, quantify the length of invasion by clicking the measure distance button, area of invasion, or other desired parameters. For a good estimation of the length of invasion, use multiple sections (2 to 4) of the same nerve.</li>
</ol>

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The authors have nothing to disclose.

In this protocol we describe an in vivo murine model of perineural invasion that allows for the quantification of sciatic nerve invasion by pancreatic cancer cells. This model enables the study of molecular mechanisms of nerve invasion. Successful experiments using this technique require a careful approach to three critical steps in the process: 1) the injection of cancer cells (steps 2.7, 2.8), 2) the extraction of invaded nerves (step 3.4), and 3) processing of harvested nerves (step 4.1).
<p class="jove_c...

Nerves form a specific tumor microenvironment that stimulates cancer growth and migration1,2,3. Perineural invasion (PNI) is the process through which cancer cells invade in and around the nerves. It may be considered as a unique route of metastasis since cancer invasion extends away from the sites of origin along nerves. PNI is found in several cancer types including pancreatic, prostate, head &#38; neck, salivary, cervical, and colorectal cancers with an incidence ranging from 22% to 100%1,2. PNI is associated with pain and correlates with poor prognosis and worse survival rates1,2.
Developing models of perineural invasion is essential to elucidate the cellular and molecular mechanisms of this process, and to test candidate therapeutic agents to decrease PNI. In vitro methods of studying interactions between cancers and nerves include the co-culture of cancer cells with nerve explants4, with dorsal root ganglions5,6,7, or with specific cells from the nerve microenvironment such as Schwann cells7. In vivo approaches, however, are more physiologically relevant, include the use of cancer mouse models in which cancer has been induced or transplanted and have the advantage of accounting for the entire nerve microenvironment. In orthotopic models of pancreatic or prostate cancer, PNI has been reported8,9,10 and the incidence of PNI may be recorded, but because of the small size of the nerves in those organs, it is difficult to see the entire nerve and therefore to quantify the extent of PNI. The model we describe here is an in vivo model of PNI in which cancer cells are injected into the sciatic nerve of mice through a simple surgical procedure11. The heterotopic transplant invades within the nerve toward the spinal cord. The length of the nerve invasion from the site of injection to the spinal cord may be measured, as well as the volume of the cancer within the nerve. Importantly, the invaded nerve can also be collected for a variety of assays including microscopic, and molecular analyses. A variety of cancer cells can be tested, and the host mice that have been genetically modified or treated with specific compounds may be used as well. This powerful assay allows for the cancer cells and the host microenvironment to be modified for investigation into the mechanisms of PNI.

<table><tbody><tr><td>Mouse</td><td></td><td></td><td>Number and age variable depending on experimental needs</td></tr><tr><td>Cell culture media (PBS, Trypsin, and DMEM+10% FBS)</td><td>Any</td><td></td><td>Steps 1.1, 1.2, 1.3.</td></tr><tr><td>Conical centrifuge tube, 50 mL</td><td>Falcon</td><td>352098</td><td>Step 1.1</td></tr><tr><td>Microcentrifuge tube 1.5 mL</td><td>Axygen</td><td>MCT-150-C-S</td><td>Step 1.2</td></tr><tr><td>Electric razor</td><td>WAHL</td><td>9962</td><td>Step 2.1. Can be substituted with commercial hair removal agent</td></tr><tr><td>Isoflurane, 250 mL</td><td>Baxter</td><td>1001936060</td><td>Step 2.2</td></tr><tr><td>Hypoallergenic surgical tape</td><td>3M Blenderm</td><td>70200419342</td><td>Step 2.3</td></tr><tr><td>Betadine Swapsticks</td><td>PDI</td><td>SKU 41350</td><td>Step 2.4</td></tr><tr><td>Webcol Alcohol Preps</td><td>Covidien</td><td>5110</td><td>Step 2.4</td></tr><tr><td>Sterile surgical tools (scissors and forceps)</td><td></td><td></td><td>Steps 2.4, 2.5, 3.3, 3.4, 3.5</td></tr><tr><td>10 &amp;mu;L Hamilton syringe</td><td>Hamilton</td><td>80308</td><td>Steps 2.7, 2.8</td></tr><tr><td>Steel Micro spatula</td><td>Fisher Scientific</td><td>S50823</td><td>Step 2.7</td></tr><tr><td>Dissecting microscope</td><td></td><td></td><td>Step 2.7</td></tr><tr><td>Bupivacine, 1 g</td><td>Enzo Life Sciences</td><td>BML-NA139-0001</td><td>Step 2.9. Reconstitute to 0.5%</td></tr><tr><td>5-0 Nylon suture</td><td>Ethicon</td><td>698H</td><td>Step 2.9</td></tr><tr><td>Tissue-Tek O.C.T. Compound</td><td>VWR</td><td>25608-930</td><td>Step 4.1</td></tr><tr><td>Tissue-Tek Cryomold Molds</td><td>VWR</td><td>25608-916</td><td>Step 4.1</td></tr></tbody></table>

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.

This method describes the surgical implantation of pancreatic cancer cells into the murine sciatic nerve to create an in vivo model of quantifiable nerve invasion.&#160;Figure 1 illustrates the anatomical location of the sciatic nerve and the site of injection. Figure 2 shows the two sciatic nerves of a nude mouse. A PBS injected nerve (left) may be compared to a nerve injected with MiaPaCa-2 cancer cells (right). The ne...

Watch this Scientific Journal Video about An In Vivo Murine Sciatic Nerve Model of Perineural Invasion at JoVE.com

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.

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 ...

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Research

JoVE Journal

Cancer Research

12.0K Views.  Memorial Sloan Kettering Cancer Center. We beschrijven een in vivo lymfkliertest model van perineural invasie door het injecteren van syngeneic pancreatic kankercellen in de nervus ischiadicus. Het model zorgt voor de kwantificering van de omvang van de zenuw invasie, en ondersteunt onderzoek naar de cellulaire en moleculaire mechanismen van perineural invasie.

Video: An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased understanding of the molecular events that give rise to glioblastomas, this cancer still remains highly refractory to conventional treatment. Surgical resection of high grade brain tumors is rarely complete due to the highly infiltrative nature of glioblastoma cells. Therapeutic approaches which attenuate glioblastoma cell invasion therefore is an attractive option. Our laboratory and others have shown that tumor associated macrophages and microglia (resident brain macrophages) strongly stimulate glioblastoma invasion. The protocol described in this paper is used to model glioblastoma-macrophage/microglia interaction using in vitro culture assays. This approach can greatly facilitate the development and/or discovery of drugs that disrupt the communication with the macrophages that enables this malignant behavior. We have established two robust coculture invasion assays where microglia/macrophages stimulate glioma cell invasion by 5 - 10 fold. Glioblastoma cells labelled with a fluorescent marker or constitutively expressing a fluorescent protein are plated without and with macrophages/microglia on matrix-coated polycarbonate chamber inserts or embedded in a three dimensional matrix. Cell invasion is assessed by using fluorescent microscopy to image and count only invasive cells on the underside of the filter. Using these assays, several pharmacological inhibitors (JNJ-28312141, PLX3397, Gefitinib, and Semapimod), have been identified which block macrophage/microglia stimulated glioblastoma invasion.

Understanding the malignant behavior of cancer requires creating accurate models of how tumor cells interact with components of the tumor microenvironment, such as macrophages. Here we describe two methods to study glioblastoma cell interaction with tumor associated macrophages and microglia where the effect on glioblastoma invasion is assessed.

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased ...

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.

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

A Mouse Model of Fatigue Induced by Peripheral Irradiation

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. Here we describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. With appropriate shielding, the irradiation targets the lower abdominal/pelvic region of the mouse, sparing the brain, in an effort to model radiation treatment received by individuals with pelvic cancers. We deliver an irradiation dose that is sufficient to induce fatigue-like behavior in mice, measured by voluntary wheel-running activity (VWRA), without causing obvious morbidity. Since wheel running is a normal, voluntary behavior in mice, its use should have little confounding effect on other behavioral tests or biological measures. Hence, wheel running can be used as a feasible outcome measure in understanding the behavioral and biological correlates of fatigue. CRF is a complex condition with frequent comorbidities, and likely has causes related both to cancer and its various treatments. The methods described in this paper are useful for investigating radiation-induced changes that contribute to the development of CRF and, more generally, to explore the biological networks that can explain the development and persistence of a peripherally-triggered but centrally-driven behavior like fatigue.

We describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. The selected non-lethal irradiation dose leads to a week-long reduction in voluntary wheel-running activity.

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

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

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

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

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

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

Neuroscience

The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer

Perineural invasion is a phenotype in which cancer surrounds or invades the nerves. It is associated with poor clinical outcome for head and neck squamous cell carcinoma and other cancers. Mechanistic studies have shown that the molecular crosstalk between nerves and tumor cells occurs prior to physical interaction. There are only a few in vivo models to study perineural invasion, especially to investigate early progression, before physical nerve-tumor interactions occur. The chick chorioallantoic membrane model has been used to study cancer invasion, because the basement membrane of the chorionic epithelium mimics that of human epithelial tissue. Here we repurposed the chick chorioallantoic membrane model to investigate perineural invasion, grafting rat dorsal root ganglia and human head and neck squamous cell carcinoma cells onto the chorionic epithelium. We have demonstrated how this model can be useful to evaluate the ability of cancer cells to invade neural tissue in vivo.&#160;

The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer

Perineural invasion is an aggressive phenotype for head and neck squamous cell carcinomas and other tumors. The chick chorioallantoic membrane model has been used for studying angiogenesis, cancer invasion, and metastasis. Here we demonstrate how this model can be utilized to assess perineural invasion in vivo.

Perineural invasion is a phenotype in which cancer surrounds or invades the nerves. It is associated with poor clinical outcome for head and neck squamous ...

Medicine

Modified Spared Nerve Injury Surgery Model of Neuropathic Pain in Mice

Spared nerve injury (SNI) is an animal model that mimics the cardinal symptoms of peripheral nerve injury for studying the molecular and cellular mechanism of neuropathic pain in mice and rats. Currently, there are two types of SNI model, one to cut and ligate the common peroneal and the tibial nerves with intact sural nerve, which is defined as SNIs in this study, and another to cut and ligate the common peroneal and the sural nerves with intact tibial nerve, which is defined as SNIt in this study. Because the sural nerve is purely sensory whereas the tibial nerve contains both motor and sensory fibers, the SNIt model has much less motor deficit than the SNIs model. In the traditional SNIt mouse model, the common peroneal and the sural nerves are cut and ligated separately. Here a modified SNIt surgery method is described to damage both common peroneal and sural nerves with only one ligation and one cut with a shorter procedure time, which is easier to perform and reduces the potential risk of stretching the sciatic or tibial nerves, and produces similar mechanical hypersensitivity as the traditional SNIt model.

The modified surgery is a simplified method for mouse or rat spared nerve injury model that requires only one ligation and one cut to injure both common peroneal and sural nerves.

Spared nerve injury (SNI) is an animal model that mimics the cardinal symptoms of peripheral nerve injury for studying the molecular and cellular ...

Establishing a Three-dimensional Co-culture Model of Murine Dorsal Root Ganglia and Cancer Cells: An In Vitro Model to Study Perineural Cancer Cell Invasion

Source: Huyett, P. et al. <a href="https://www.jove.com/video/55043" target="_blank">A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma.</a> J. Vis. Exp. (2017)
In this video, we demonstrate the procedure for developing an in vitro perineural cancer cell invasion model by co-culturing mouse dorsal root ganglion and neurotropic cancer cells. The model can be utilized to conduct cancer assays, producing rapid and reproducible results.

Source: Huyett, P. et al. A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma. J. Vis. Exp. (2017)
In this video, we demonstrate the ...

JoVE Encyclopedia of Experiments

Head and Neck Cancer

Assays & techniques

Murine Sciatic Nerve Model of Perineural Invasion or PNI: Extraction and Processing of Sciatic Nerve to Understand Invasion by Cancer Cells

Source: Deborde, S. et al. <a href="https://www.jove.com/video/56857" target="_blank">An In Vivo Murine Sciatic Nerve Model of Perineural Invasion.</a> J. Vis. Exp. (2018)
In this video, we describe the procedure to establish a murine model of perineural invasion by injecting cancer cells into the sciatic nerve. The model can help gain an understanding of the magnitude of nerve invasion for subsequent assessment of anti-cancer drugs.

Source: Deborde, S. et al. An In Vivo Murine Sciatic Nerve Model of Perineural Invasion. J. Vis. Exp. (2018)
In this video, we describe the procedure to ...

Cancers of the Nervous System

Ex vivo studies

Murine Perineural Invasion Model: Generating an In Vivo Model to Simulate Perineural Invasion in Mouse Sciatic Nerve

Source: Deborde, S. et al. <a href="https://www.jove.com/video/56857" target="_blank">An In Vivo Murine Sciatic Nerve Model of Perineural Invasion.</a> J. Vis. Exp. (2018)
In this video, we develop an in vivo mouse model for perineural invasion by injecting pancreatic cancer cells into the sciatic nerve. This model helps quantify the extent of invasion and study the interactions between cancer cells and nerves.

Source: Deborde, S. et al. An In Vivo Murine Sciatic Nerve Model of Perineural Invasion. J. Vis. Exp. (2018)
In this video, we develop an in vivo mouse ...

In vivo studies

Establishing a Three-dimensional Co-culture Model of Murine Dorsal Root Ganglia and Cancer Cells: An In Vitro Model To Study Perineural Cancer Cell Invasion

To prepare the semi-solid matrix droplets, transfer one glass well-bottom plate from ice onto an ice block under the operating microscope. Place the tip of a no more than 10-microliter pipette directly onto the glass at a 45-degree angle and carefully dispense a 1.5-microliter droplet of matrix onto the glass. Slowly move the pipette tip away from the glass as the matrix becomes engaged with the plate.
Deposit one droplet of matrix onto each of the four corners of the plate and transfer the plate to a room temperature surface for about 1 minute. When the matrix has slightly stiffened, return the plate to the ice block and use closed microscopic forceps to scoop the DRG gently with the left hand.
Using the right hand, carefully transfer the nerve tissue to the tip of a 21G needle and use the needle to gently insert the DRG into the middle of the matrix droplet. The DRG should release easily into the matrix for central positioning with the needle.
After inserting a DRG piece into each of the four droplets, place the plate into a 37 degrees Celsius incubator for 3 minutes. When all of the DRG have been embedded, hold the plates one at a time at a slight angle and slowly add 4 milliliters of DMEM supplemented with 10% FBS to the plates such that the medium only gradually comes into contact with the matrix-DRG units.
Return the plates to the incubator for another 48 to 72 hours. When the neurites reach at least 75% of the way to the edge of the matrix, aspirate the medium from the wells without removing the matrix-DRG units. Then, using a 200-microliter pipette with a smooth trigger, place two drops of 3 times 10 to the fifth cells per milliliter of medium over each matrix DRG assay and return the plates to the incubator. After one hour, gently add 4 milliliters of DMEM with 10% FBS along the sidewall of the glass-bottom plate and return the plates to the incubator until their next microscopic evaluation.

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

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