THE INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC)
IACUC / LARC STANDARD PROCEDURES
RETRO-ORBITAL INJECTION IN MICE
Description of procedure:
The retro-orbital injection is an acceptable alternative to the tail vein injection route, if technicians
receive proper training. The route designated by the protocol should therefore be based on the
researcher’s level of training and proficiency with the retro-orbital or tail vein injection.
When administering a retro-orbital injection, anesthesia is required. When performed proficiently,
this procedure will take less than five minutes. In which case, a heat source is not required.
The injectable solution is limited to 200µL. Cell suspensions are filtered or agitated prior to
injection to prevent cell clumping. A mouse receives no more than one injection per day. When
more than one injection is required, alternate between eyes and allow 1-2 days between injection
sites. Do not exceed two injections per eye in a mouse.
Supplies:




Anesthesia
Gloves
Needle (needle not to exceed ½” to prevent trauma)
Gauze sponges
1. Anesthetize the mouse.
2. Position the mouse on its side. Restrain it with the thumb and middle finger of the nondominant hand, pulling back the loose skin over the shoulders and behind the ears.
3. Use the index finger of the non-dominant hand to draw back the skin above the eye and
the thumb to draw back the skin below the eye. The eye will protrude slightly.
4. Insert the needle approximately at a 45° angle to the eye, lateral to the medial canthus,
through the conjunctival membrane. There is a degree of resistance, which causes the
eye to retreat back into the sinus, until the needle pierces through the conjunctiva. The
needle will be positioned behind the globe of the eye in the retrobulbar sinus. A sharp
cutting needle is preferred as it results in reduced tissue distortion and damage.
5.
6.
7.
8.
9.
10.
Inject into the retrobulbar sinus.
Remove needle gently to prevent injury to the eye.
Close the eyelid and apply mild pressure to the injection site with a gauze sponge.
Monitor the mouse during the recovery process.
Examine the injection site for swelling or other visible trauma.
If no adverse effects are observed, return the mouse to its home cage, once it regains its
righting reflex.
Updated June/2010
Please contact IACUC Training and Compliance at IACUCTrainer@ucsf.edu for training.
Literature search words required:
Literature search was performed for refinement of this Standard Procedure on May 26, 2010:
Key Words
Retro orbital injection
Retro orbital tail vein comparison
Retro orbital injection complications
Search Site
Pubmed
PubMed
Years Covered
Full database
up to 2010
Full database
up to 2010
Agents:
This procedure requires inhalant anesthesia (isoflurane).
All agents administered to animals should be listed in the “Agents” section of RIO.
Adverse Effects:
Procedure, Agent or
Phenotype
Incorrect placement of the
needle
Tumor cells injection
Monitoring Parameters
General appearance and signs
of trauma around injection site
Grooming habits, skin color,
lethargy, general
appearance/activity level
Adverse Effects
Potential Adverse
Effects
Swelling around injection
site, proptosis of the eye or
eye trauma
Tumor growth around
injection site/behind the
eye
Monitoring Parameters
Frequency
During and after procedure,
following day and prior to
next injection.
During and after procedure,
following day and prior to
next injection.
Management
Consult a LARC veterinarian
or euthanize.
Consult a LARC veterinarian
or euthanize. Change
injection site for the other
animals in the study (e.g. tail
vein injection).
PI/Lab will Document
Will document only if
problem is identified.
Will document only if
problem is identified.
Describe the conditions, complications, and criteria (e.g. uncontrolled infection, loss
of more than 15% body weight, etc.) that would lead to removal of an animal from the
study, and describe how this will be accomplished (e.g. stopping treatment,
euthanasia).
Euthanize if untreatable or permanent eye trauma occurs at the injection site or uncontrolled
bleeding is observed in the mouse. In addition, if injecting tumor cells and the tumor rapidly
grows around injection site/behind the eye, euthanize the animal.
For all investigators housing animals with tumor formation, skin lesions, neurological
deficits, or that are in Category E, list the expected characteristics/clinical
presentations and endpoints of the animal model and the criteria for euthanasia. Note:
The IACUC also requires such lists to be posted in the respective animal rooms and
monitored by the IACUC compliance staff and LARC, to assure PI adherence to the
endpoints listed.
Updated June/2010
Updated June/2010