Template for Rodent Retro orbital Bleeding

Purpose

Blood withdrawal from the retro orbital sinus can be used in both rats and mice (although it is usually not a method of choice in the rat) by penetrating the retro-orbital plexus/sinus with a glass capillary tube or Pasteur pipette. The NIH ARAC has determined that in the hands of a skilled operator retro-orbital bleeding is a humane procedure that produces minimal and transient pain/distress. This procedure is relatively rapid enabling the collection of blood from a large number of animals within a short period of time and the volume of blood obtainable per animal is medium to large. Retro-orbital Sampling: provides good sample quality. Potential contamination with topical anesthetic, if used, should be taken into account.

Guidelines

• Both the quantity and frequency of blood sampling is dependent on the circulating blood volume of the animal. The approximate blood volume

  is 1.5 ml for a 20 gram mouse and 13 ml for a 200 gram rat. In general, no more than 15% of the animal's blood volume should be removed at one sampling. This is approximately equal to 1% of body weight. Sampling of this amount should not occur more than once every two weeks. Volumes greater than 15% should be justified and approved by the IACUC and appropriate fluid replacement considered.

  •  Retro orbital blood sampling is not amenable to frequent repeated sampling from the same orbit (2 weeks recommended between successive bleeds).

  • In the hands of an unskilled operator, retro-orbital sampling has a greater potential than other blood collection routes to result in complications.

  •  The presence of a plexus rather than sinus in the rat can lead to greater orbital tissue damage than in the mouse.

  •  Retro-orbital bleeding is ideally conducted under general anesthesia in rats and mice although it can be performed in awake mice when justified and approved by the IACUC. A topical ophthalmic anesthetic should be applied prior to the procedure in nonanesthetized mice.

  •  Due to restraint issues retro-orbital sampling in the rat should only be conducted under general anesthesia.

  •  In both mice and rats, care must be taken to ensure adequate hemostasis following the procedure.

Template

Animals are anesthetized with ___________________. When the animal is no longer moving voluntarily it is placed on a paper towel The forefinger of the operator's nondominant hand is used to pull the facial skin taut and cause the eyes to protrude slightly while the skin at the back of the neck is grasped by the thumb and remaining fingers to restrain. Breathing and color are monitored throughout the procedure to ensure that the restraint does not comprimise the airway. Using the dominant hand, the tip of capillary tube or pipette is gently inserted below the eye at approximately a 45 degree angle into the space between the globe and the lower eyelid. When the tip of the pipette contacts the boney floor of the orbit it is gently twisted between thumb and forefinger to rupture the capillary plexus/sinus. Blood is allowed to flow by capillary action into the pipette. Care is taken not to take any more blood than is needed. At the conclusion of the blood withdrawal tension on the animal is released and a gauze pad is gently pressed over the eye for a few seconds until the bleeding has stopped. Normal color and respiration are reconfirmed and the animal is returned to its cage for recovery . Alternate eyes are used for successive bleeds. A maximum of two bleeds per eye (four bleeds total) will be done.

References

1. Perspectives on Animal Use Biological Effects of Blood Loss: Implications for Sampling Volumes and Techniques. In: ILAR News (1989), 31(4).
2. Removal of blood from laboratory mammals and birds: First report of the BVA/FRAME/RSPCA/UFAW Joint working group on refinement. Lab Anim (1993) 27, 1-22.
3. J. Donovan and P. Brown. Blood Collection. In: Current Protocols in Immunology, (eds: A. Kruisbeek et al), John Wiley & Sons, New York , NY , USA , Unit 1.7.
4. H. van Herck et al., Orbital sinus blood sampling in rats as performed by different technicians: the influence of technique and expertise. Lab Anim (1998) 32, 377-386.
5. http://www.eslav.org/efpia.htm
6. http://www.uib.no/vivariet/mou_blood/Blood_coll_mice_.html
7. Public Health Service Policy on Humane Care and Use of Laboratory Animals , Reprint, October, 2000.
8. Fox, J.G., Cohen, B.J., and Loew, F.M., Laboratory Animal Medicine . Academic Press, Inc., Orlando , FL , 1984.
9. Greenstein Survey on Retro Orbital Sinus Bleeding in Mice, June 6, 1996 .
10. Evaluation of Proparacaine Hydrochloride as a Topical Anesthetic for the Collections of Blood from the Orbital Sinus of Mice.
11. Terril-Robb, L.A. , Suckow, M.A., Grigdesby, C.F., Contemporary Topics , vol. 35, pg. 13, 1996.