In this video, intra cerebro ventricular and intravenous injection methods will be demonstrated in neonatal mice. For intra cerebro ventricular injection, the anesthetized mouse is first placed on top of a fiber optic light source to illuminate the sagittal and transfer sinuses. The inoculum is then carefully injected at a point lateral to the sagittal suture and rostral to the coronary suture.
If performed correctly, the inoculum will be distributed first in the injected ventricle and will eventually reach the other ventricle for intravenous injection. The mouse is secured to a transluminator in such a way that the facial vein is exposed. The inoculum is then slowly injected directly in the vein and becomes distributed systemically.
The implications of these techniques extend toward therapy of many diseases and disorders because they allow for the optimal placement of therapeutics. The ICV technique provides a means to cross the blood-brain barrier, while the IV technique allows for systemic therapeutic delivery. First, prepare the injection solution in a micro centrifuge tube.
In this example, the inoculums contain triam blue for better visualization of the injection site. To inject mice use a sterilized and calibrated glass micropipet needle attached to a series of plastic tubing, gradually increasing in diameter and ending in a three milliliter plastic syringe. Using flat end tweezers, break off the tip of the glass needle so that it's three millimeters long.
This will allow at least two millimeter penetration into the brain. Carefully load the solution to be injected into the glass micropipet needle by placing it diagonally in the micro centrifuge tube and pulling on the syringe plunger. Stop pulling the plunger as soon as the needle loading is completed.
To avoid contaminating the tubing, remove the syringe from the tubing system attached to the glass micro pipet. Extend the plunger completely and reattach the needle firmly. Hold the micro pipee glass needle between the thumb and index finger.
Place the syringe between the ring and little fingers with a plunger touching the palm of the injecting hand cryo anesthetize the two day old mouse. Then firmly grasp the cry anesthetized mouse by the skin at the base of the head, and place it on a fiber optic light source to illuminate the superior sagittal and transfer sinuses. The injection site is located about a quarter of a millimeter laterally to either side of the confluence of these sinuses holding the syringe perpendicular to the skull surface.
Insert the needle penetrating two millimeters deep. Using the palm of the injecting hand, slowly push the plunger down to inject the solution. Wait 15 seconds before removing the needle to prevent backflow and monitor the mouse for cranial swelling or signs of ruptured vessels.
Once the mouse has been injected, place it in a prewarm container for five to 10 minutes to allow it to recover. Compare the injected mouse to a non injected mouse. 10 to 15 minutes after intra cerebro ventricular injection of inoculum containing triam blue.
The fluid should be distributed in the injected side of the brain after 60 minutes due to the connectivity of the cerebral ventricles, uniform distribution of triam blue in both cerebral hemispheres and olfactory bulbs should be visible. Finally, after 12 hours, the inoculum will be visible in the rostral central spinal canal. Prepare the injection solution and add green food dye at a one to 100 dilution.
To visualize the injection, attach a small plastic lure to the end of a 100 microliter glass Hamilton syringe. Fit the syringe with a 33 gauge 0.25 inch hypodermic needle and ensure that all parts are securely connected. Load the syringe with the volume to be injected.
Be sure to remove any air bubbles from the inoculum as injecting air intravenously is lethal. Use surgical tape and gauze to secure the neonate to the transluminator. By first securing the forearm and body to the transluminator and then securing the head so that the facial vein is visible.
Ensure that the tape is loose enough that the animal can still breathe. Using a 2.25 x headman magnifier to allow better visualization, slowly insert the needle into the vein. Because the vein is superficial, the needle will remain visible under the skin.
Slowly infuse the inoculum into the vein. Wait 15 seconds before removing the needle to allow all fluid to be expelled. Once the needle has been removed, use a piece of gauze to apply pressure to the injection site until the bleeding stops.
Return the neonate to a warm place and allow five minutes for it to recover. Monitor it for signs of distress. If the injection has been performed correctly, the animal should not show signs of discomfort.
Once the mouse is recovered, return it to its cage. If the intravenous injection has been performed correctly, the neonate will become green. After watching this video, you should have a good understanding of how to administer ICV and IV injections in neonatal mice.
These techniques can be used as a means of injecting the therapeutic agent of your choice.
