Intracranial Orthotopic Model: A Procedure to Implant Cancer Cells in Mouse Brain

Protokol

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

1. Injecting Cancer Cells into the Brain

1. Place an anesthetized mouse with an exposed calvarial window on the stereotatic frame.
2. Attach an automatic injector unit to the stereotactic apparatus (Figure 1).
3. Pull up >2 µL of cells thoroughly resuspended in 1x DPBS in a clean Hamilton syringe. Be sure to resuspend cells immediately prior to filling the syringe to reduce clump formation and ensure a homogenous cell slurry. It is ideal to pull up 6–8µL of cell volume to ensure there are no air pockets/bubbles.
4. Place the Hamilton syringe onto the injector apparatus, and prime the needle for injection by dispensing a small amount of volume onto a disposable sterile drape to ensure the injector is working properly. Wipe the syringe with 70% ethanol with a cotton tip applicator (Figure 1, inset). This removes tumor cells from the outer barrel of the needle shaft reducing the risk of tumor cell seeding along the injection tract.
5. Align the needle tip to the center of the calvarial window, nearly touching the exposed cerebrum.
6. Reset the digital Vernier scale to zero.
7. Slowly insert the needle to a depth of 3 mm into the brain and allow the needle to remain in the brain for at least 60 seconds before proceeding. This time frame allows the brain parenchyma to conform around the needle, which reduces back pressure and potential expulsion of tumor cells along the needle tract.
8. Select Run on the injector screen to begin the delivery of cells to the injection site. It will take approximately 5 min to inject this volume. The prolonged time for this step is to reduce secondary damage caused by injection force on the brain parenchyma.
9. Once the injection protocol is finished, allow the needle to rest in the brain for at least 3 min, again allowing the brain parenchyma to acclimate to the injection.
10. After at least 3 min, raise the needle from the brain at a rate of 0.75 mm/min. Do this at an extremely slow and consistent manner to reduce back pressure and tumor tracking up the needle tract.
11. Once the needle has exited the brain, carefully remove the Hamilton syringe from injector.
12. Apply warm bone wax to the calvarial window using a sterile cotton swab. The bone wax acts as a physical barrier to keep the tumor within the skull.
13. Close the incision (e.g., 5-0 PDS dissolvable sutures in a simple interrupted pattern or suture clips, whichever is most comfortable for the surgeon).
14. Stop the administration of anesthesia and remove the mouse from the apparatus by unlocking and sliding out the ear bars, sliding the nose cone off the mouse, and disengaging the teeth from the mouth bar.
15. Place the mouse in a clean cage that is on a warmer set to 37 °C for recovery. Monitor mice during recovery, which typically occurs 10–15 minutes after anesthesia has been discontinued.

Sonuçlar

Figure 1: Stereotactic and anesthesia systems. Stereotactic stand with automated syringe pump and mouse elevating box. The box is an inverted pipette tip box containing hand warmers used to elevate the mouse to the appropriate height and maintain body temperature. Inset shows the orientation of the Hamilton syringe in the automated injection apparatus.

Malzemeler

Name	Company	Catalog Number	Comments
Bone Wax	Surgical Specialities	903	Sterile and malleable beeswax and isopropyl palmitate
Cotton tip applicators	Puritan	25-806 10WC	Sterile long stemmed cotton tip applicators
Suture	Covidien	SP5686G	45 cm swedged 5-0 monofilament polypropylene suture
Skin Glue	Vetbond	1469SB	Skin safe wounds adhesive
Stereotaxic Unit
Mouse Gas Anesthesia Head Holder	Kopf	Model 923-B	Mouth bar with teeth hole and nosecone
Non-Rupture Ear Bars	Kopf	Model 922	Ear bars suitable for mouse applications
Stereotaxic Instrument	Kopf	Model 940	Base plate, frame and linear scale assembly with digital readout monitor
Injector
Injector Needle and syringe	Hamilton	80366	26 gauge needle, 51 mm needle length and 10 μL volume syringe
Legato 130A automated Syringe Pump	KD Scientific	P/N: 788130	Programmable touch screen base with automated injector
Anesthesia Machine
SomnoSuite Low-Flow Digital Vaporizer	Kent Scientific	SS-01	Digital anesthesia machine