brain metastasis mouse model: a method to generate brain metastasis mouse model by intracarotid cancer cell injection

Brain Metastasis Mouse Model: A Method to Generate Brain Metastasis Mouse Model by Intracarotid Cancer Cell Injection

In this procedure, anesthetize the mouse with ketamine/xylazine cocktail by intraperitoneal injection. Confirm complete anesthesia by pinching the animal&#39;s feet and observe the lack of response. Next, remove the neck hair by shaving or using a small amount of hair removal product.
Then, place the mouse on a glass plate and secure with rubber bands. Clean the skin by applying 70% alcohol and povidone-iodine. Now, make an incision in the skin about 1 centimeter long with a surgical scalpel. Then, bluntly dissect the muscle with surgical forceps to expose the carotid artery underneath.
Separate the carotid artery from the adjacent vagus nerve with surgical forceps. Afterward, place two sutures - one distal and the other proximal to the cotton ball - and make loose knots. Tighten the proximal knot to block the blood flow into the injection site.
After that, moisten a small cotton ball with buffer and place it underneath the carotid artery of the intended site of injection. Then, proceed to cancer cell injection if the carotid artery on the cotton ball appears fully pressurized with fresh red blood.
In this step, vortex and draw 100 microliters of cancer cells into the syringe. Under the dissecting microscope, slowly insert the 31-gauge needle with bevel up into the lumen of the carotid artery sitting on top of the cotton ball. Slowly inject the cells from the syringe into the carotid artery.
Successful injection can be observed by the changing color of nearby blood vessels and musculature when the buffered cancer cells are pushed into the circulation. When the injection is complete, lift the distal ligature gently to prevent regurgitation. Then, quickly tighten the distal knot to complete the injection process.
After completing the injection, tighten the ligatures and trim the excess silk suture with micro-scissors. Move back the separated muscle to cover the wound site and close the skin with two staples.
For recovery, transfer the animal onto a heating pad. Then, administer buprenorphine via subcutaneous injection as post-surgical analgesia. Once it regains consciousness and resumes normal behavior, return the mouse to its housing location. Provide the animal with gel food for several days post-surgery.

brain-metastasis-mouse-model-method-to-generate-brain-metastasis

Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Cancer Research

Cancers of the Nervous System

In vivo studies

EoE Sub Articles

eoe sub articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Prepare Mice for Tumor Cell Injection

<ol>
	<li>Anesthetize a mouse by intraperitoneal (i.p.) injection of ketamine/xylazine cocktail (ketamine 100 mg/kg, xylazine 10 mg/kg).</li>
	<li>Confirm complete anesthesia by pinching the animal&#39;s feet and observe lack of response.</li>
	<li>Place the mouse on a glass plate (used for casting protein gels) and secure with rubber bands.</li>
	<li>If necessary, remove the hair of neck by shaving or applying a small amount of hair removal product and wiping the hair off with paper towel. 
	NOTE: If using nude mice, skip this step as they do not have body hair.</li>
	<li value="5">Clean the neck skin by applying povidone-iodine and 70% alcohol.</li>
	<li>Place mouse on stage of the dissecting microscope</li>
	<li>Make an incision in the skin ~1 cm long with a surgical scalpel.</li>
	<li>Bluntly dissect the muscle with toothed forceps to expose the carotid artery underneath.</li>
	<li>Separate the carotid artery from the adjacent vagus nerve with surgical forceps.</li>
	<li>Using surgical forceps, separate some cotton from a cotton ball, moisten, and fashion into a smaller cotton ball. Place this buffer-moisturized cotton ball underneath the carotid artery of intended site of injection.</li>
	<li>Place a suture distal and proximal to the cotton ball and make loose knots. Tighten the proximal knot to block the blood flow into the injection site.</li>
	<li>Proceed to cancer cell injection if the carotid artery on the cotton ball appears fully pressurized with fresh red blood.</li>
</ol>

2. Cancer Cell Injection into Carotid Artery

<ol>
	<li>Vortex and draw 100 &#181;L of cancer cells into the syringe.</li>
	<li>Under the dissecting microscope, slowly insert the 31G needle (with bevel up) into the lumen of the carotid artery sitting on top of the cotton ball.</li>
	<li>Slowly inject the cells from the syringe into the carotid artery. Successful injection can be observed under the microscope by the changing color of nearby blood vessels and musculature when buffered cancer cells are pushed into the circulation.</li>
	<li>When injection of the entire volume is complete, lift the distal carotid artery gently to prevent regurgitation.</li>
	<li>Quickly tighten the distal knot to complete the injection process.</li>
	<li>After completing the injection, tighten the ligatures and trim the excess silk suture with micro-scissors under the dissecting microscope. Move back the separated muscle to cover the wound site. Close the skin with two staples.</li>
</ol>

3. Surgical Recovery

<ol>
	<li>Move the operated animal onto a heating pad for recovery. It may take 30&#8211;60 min for the animals to regain consciousness. In the meantime, administer buprenorphine (0.05 mg/kg)&#160;via&#160;subcutaneous injection as post-surgical analgesia. &#160; 
	NOTE: Analgesia should be provided according to your&#160;local IACUC-approved protocol.&#160;</li>
	<li value="2">Once the animals awaken and resume their normal behavior, return the mice to their housing location. Provide mice with gel food for several days post-surgery. Maintain a surgical record and keep it in the room.</li>
</ol>

4. Non-invasive&#160;In Vivo&#160;Imaging

<ol>
	<li>For cells labeled with luciferase reporter constructs, measure cancer cell injection efficiency using&#160;in vivo&#160;imaging system.&#160;&#160; 
	NOTE: Imaging is not performed on the day of injection to avoid excessive stress to research animals.</li>
</ol>

<table><tbody><tr><td>Ketamine hydrochloride/xylazine hydrochloride solution</td><td>Sigma-Aldrich</td><td>K113</td><td></td></tr><tr><td>Routine Stereomicroscope Leica M50</td><td>Leica</td><td>M50</td><td>The microscope is modular and highly configurable to fit particular space requirements</td></tr><tr><td>Surgical disposable scapel</td><td>Integra Miltex</td><td>4-410</td><td>To make skin incisions</td></tr><tr><td>Tissue Forceps - 1 x 2 Teeth</td><td>Fine Science Tools</td><td>11021-12</td><td>To bluntly dissect muscles</td></tr><tr><td>Dumont #5 - Mirror Finish Forceps</td><td>Fine Science Tools</td><td>11252-23</td><td>To separate and prepare the carotid artery for injection</td></tr><tr><td>Spring Scissors</td><td>Fine Science Tools</td><td>15025-10</td><td>To cut sutures</td></tr><tr><td>EZ Clip Kit</td><td>Stoelting</td><td>59020</td><td>For wound closure</td></tr><tr><td>BD Insulin Syringe</td><td>Becton Dickinson</td><td>328438</td><td>For cell injection</td></tr><tr><td>IVIS Spectrum&amp;nbsp;In Vivo&amp;nbsp;Imaging System</td><td>PerkinElmer</td><td>124262</td><td></td></tr></tbody></table>

Source: Zhang, C. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/55085" target="_blank">Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis.</a> J. Vis. Exp. (2017)
This video demonstrates the establishment of a brain metastasis mouse model by intracarotid injection of fluorescently labeled cancer cells. The mouse model can be used to study the effects of novel therapeutics and the underlying physiological mechanisms.

Source: Zhang, C. et al. Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis. J. Vis. Exp. (2017)
This video demonstrates the ...

In brain metastasis, cancer cells from other affected organs, or the primary site of origin, spread into the central nervous system to form secondary tumors. To generate a mouse brain metastasis model, prep an anesthetized mouse in the supine position. Make a small incision in the neck region.
Dissect the muscles to expose one of the underlying carotid arteries. Carotid arteries are a pair of major blood vessels in the neck, supplying blood to the brain and head. Separate the carotid artery from the adjacent vagus nerve. Next, place suture knots around the proximal and distal ends of the artery.
Tighten the proximal knot to obstruct the blood flow into the target injection site. Then, place a buffer-soaked cotton ball under the target injection site to immobilize and stretch the artery for additional support. Take a syringe filled with green fluorescent protein or GFP-labeled cancer cell suspension.
Gently inject the cell suspension into the lumen of the carotid artery. Retract the needle and tighten the distal knot to prevent leakage of cancer cells. Lastly, suture the incision. Allow the mouse to recover. Over time, cancer cells begin to migrate and form secondary tumors in the brain, confirming the successful establishment of a brain metastasis model.

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Brain Metastasis Mouse Model: A Method to Generate Brain Metastasis Mouse Model by Intracarotid Cancer Cell Injection

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Brain Metastasis Mouse Model: A Method to Generate Brain Metastasis Mouse Model by Intracarotid Cancer Cell Injection