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.
The procedure is facilitated by being seated at a lab table, bench,
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
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).