FILARIASIS RESEARCH REAGENT RESOURCE CENTER:
University of Wisconsin Oshkosh
STANDARD OPERATING PROCEDURE
TITLE: Retro-orbital Eye Bleeds of Gerbils and Hamsters
SOP NUMBER: A. vit. 4
1. PURPOSE
This standard operating procedure outlines the collection of peripheral blood samples by retro-orbital eye
bleed from hamsters and gerbils.
2. MATERIALS
2.1. Anesthesia of choice
Note: The same anesthesia should be used for each blood collection that is intended for
microfilariae (mf) enumeration or tick infection, as the anesthesia used has been found to affect
the concentration of mf in the peripheral blood. Additionally, the blood draw site can affect mf
concentration. Therefore, the same site should be used for each blood collection if you wish to
track mf counts over time. For example, mf counts of blood collected retro-orbitally are
sometimes very different than blood collected from the saphenous vein.
2.2. Hamsters and/or gerbils of which you wish to evaluate microfilaremia. Gerbils and hamsters are
expected to be microfilaremic 50-60 days post infection.
2.3. Heparinized sterile 70 ul capillary tubes (Fisher Scientific Cat #: 22-260-950)
2.4. Cotton balls
2.5. 1.5 mL Microfuge tubes
2.6. Sterile 0.5% proparacaine hydrochloride ophthalmic solution
3. PROCEDURES
3.1. Anesthetize the animal with isoflurane or anesthesia of choice.
For gerbils: Use 5% isoflurane to induce anesthesia and 2.5-3.5% isoflurane for maintenance
anesthesia during the procedure. Isoflurane is used as a mixture with oxygen (100%) at a flow
rate of 1 L/min.
Last revision: 5/27/13 SS & LM
A. vit. 4
For hamsters: Use 4% isoflurane to induce anesthesia and 2.5-3.0% isoflurane for maintenance
anesthesia during the procedure. Isoflurane is used as a mixture with oxygen (100%) at a flow
rate of 1 L/min.
3.2. It is usually easiest for people to draw blood from the same eye of the animal as their dominant
hand (i.e., if you are right handed, you will probably find it easiest to draw blood from the right
eye of the animal). It is recommended that you alternate eyes each blood draw to allow for
proper healing of tissues and to reduce the amount of scar tissue in each eye.
Insert the tip of the capillary tube at the medial canthus (inside corner) of the eye. Ensure that
the capillary tube is under the nictitating membrane (third eyelid) and that the bottom eyelid is
not rolled medially (hair touching the eyeball) when inserting the capillary tube.
3.3. With the capillary tube under the bottom eyelid, slide it caudally ~2/3 the length of the eyeball
toward the lateral canthus. Do not rub the tube against the eyeball.
3.4. With gentle force, push the capillary tube deeper into the tissue under the eye, being careful not
to puncture the globe (eyeball) itself. Also, do not accidently use the capillary tube as a lever
and force the globe out of its socket.
Pushing the capillary tube into this deeper tissue might be enough for blood to enter the tube by
capillary action. Retracting the tube a few millimeters might help facilitate blood flow.
If blood is not flowing, point the capillary tube down a few millimeters toward the ventral aspect
of the orbital bone and grind the tube against the bone. This will often break enough capillaries
to allow for blood collection. Here too, retracting the tube a few millimeters might help facilitate
blood flow. It can also be helpful to ensure the free end of the capillary tube is lower than the
animal by lifting and rotating the animal, thereby pointing the free end of the tube down.
3.5. Blood can be collected into a microfuge tube if the volume collected in the capillary tube is
insufficient.
It is not recommended to collect more than 250 µl from each animal. At least two weeks must
pass before another blood sample is taken from the same animal.
3.6. When enough blood has been collected, remove the capillary tube and apply light pressure to the
eye with a sterile cotton ball moistened with water to stop the flow of blood.
3.7. When bleeding has stopped, wipe away any excess blood with the cotton ball.
3.8. Apply one drop of 0.5% proparacaine hydrochloride ophthalmic solution to the bled eye.
3.9. Monitor the animal(s) closely during recovery from anesthesia.
3.10. The gerbil(s) should be given black oil sunflower seeds or a similar high calorie diet following
blood collection.
3.11. Time to Completion: 10-15 minutes per animal
Last revision: 5/27/13 SS & LM
A. vit. 4