Background:
Retro-orbital injections work very well for transplants of stem cells as an alternative to tail vein intravenous injections. They are much less challenging technically than tail vein injections, and anyone who is skilled at retro-orbital blood sampling can master the technique almost immediately. Non-anesthetized mice can be transplanted with only hand restraint in only a matter of seconds with no need for warming or other pre-transplant preparation.

Procedure:
The procedure is facilitated by being seated at a lab table, bench, or hood.

Pick up the mouse with the left hand (for right-handers) and hold it securely by thumb and third finger on the nape of the neck, with the tail wrapped around the little finger.

Place the mouse against absorbent paper on the table surface, keeping it motionless. Using the index finger, gently retract the fur above the eye, similar to a bleeding procedure.

Using a 1 cc syringe and a 27 or 30g needle, with the bevel facing outward insert the needle at a 45° angle into the center of the area where you would normally insert a bleeding tube. Carefully introduce the tip of the needle to penetrate the retro-orbital sinus. Make sure the needle is approximately mid-sinus. Inject up to 200 µl slowly and release gently so that the mouse stays relaxed. It is imperative that the material to be injected contains no clumped material (e.g., always filter cell suspension prior to injection). After injection, the needle should be carefully removed, keeping the bevel outward to protect the mouse’s eye from being scratched.

For multiple injections, at least 2 days should elapse between injections. Eyes should be alternated for subsequent injections, with a maximum of 2 injections per eye per mouse. The use of Tetracaine topical anesthetic one minute prior to injection is recommended.

It should be noted that retro-orbital injection is significantly less traumatic than eye bleeding at the same site, yet eye trauma is still likely to occur and the site should be inspected or observed post-procedure. No injections may be performed in a damaged eye. In the event that both eyes are damaged and/or become opaque, eye injections must cease and DLAR should be consulted for treatment options.

It is advisable to practice first on anesthetized mice, but after mastery of the technique, anesthesia should not be necessary. Following anesthesia, the animal(s) should be observed until they become conscious and ambulatory, after which they will be returned in their cage(s) to the cage racks. As with all procedures, DLAR staff will determine whether an individual is adequately skilled to carry out the technique.

Note of caution: material injected by this route directly enters the blood system. For instance, anesthesia delivered by this route will act very quickly and must be dosed appropriately (for example, much less than an equivalent i.p. dose).