Retro-orbital

Technique

Also referred to as peri-orbital, posterior-orbital and orbital venous plexus bleeding.

Retro-orbital bleeding should typically be performed as a terminal procedure. It should only be used with recovery in rare circumstances with exceptional scientific justification (e.g. where a large blood volume is necessary or where peripheral veins are used for dosing), because of its potential impact on animal welfare. Adverse effects reported for this technique include (1-2% of animals):

  • Retro-orbital haemorrhage resulting in haematoma and excessive pressure on the eye
  • Corneal ulceration, keratitis, pannus formation, rupture of the globe and micro-ophthalmia caused by proptosis of the globe
  • Damage to the optic nerve and other intra-orbital structures which can lead to deficits in vision and even blindness
  • Fracture of the fragile bones of the orbit and neural damage by the micro-pipette
  • Penetration of the eye globe itself with a loss of vitreous humour
  • Necrotic dacryoadenitis of the Harderian gland

Where its use is unavoidable, retro-orbital bleeding should only be used under general anaesthesia. Because of the severity of the adverse effects that can occur with this technique, even in skilled hands, it is essential that it is conducted only by staff members competent (practiced) in the technique.

Blood is collected from the venous plexus. The presence of a plexus rather than a sinus in the rat can lead to greater orbital tissue damage than in the mouse. The rat is restrained, the neck gently scruffed and the eye made to bulge. A capillary tube/pipette is inserted medially, laterally or dorsally. Blood is allowed to flow by capillary action into the capillary tube/pipette. The sample obtained is a mixture of venous blood and tissue fluid, and is not representative of venous blood.

Blood flow can be stopped by applying gentle finger pressure to the soft tissue. A finger should be placed over the closed eyelid for approximately 30 seconds. The rat should be checked for post-operative peri-orbital lesions approximately 30 minutes after blood sampling and on at least one more occasion within two hours of the sampling.

Sequential sampling is not recommended as histological changes, abnormal clinical signs and evidence of discomfort have been reported. Ideally, no more than one bleed per eye should be taken - where a second retro-orbital bleed is required this should be performed as a terminal procedure. In rare circumstances where there is exceptional scientific justification sequential sampling may be used. In such instances only one orbit should be sampled from at any one sampling time. Adequate time should elapse between sampling to allow peri-orbital tissue repair to take place. An interval of two weeks between bleeds should allow damaged tissue to repair in most cases.

Summary

Number of samples It is recommended that only one sample be taken.
Sample volume Up to 4 ml with recovery; 4-10 ml non-recovery.
Equipment A glass capillary tube or Pasteur pipette.
Staff resource One person is required to take the blood sample.
Other Careful monitoring for adverse effects is necessary peri-operatively.
Adverse effects See above.

Resources and references

  • A good practice guide to the administration of substances and removal of blood, including routes and volumes.
  • Brown C (2006), Blood collection from the tail of a rat. Lab Animal Europe. 6(8) 35-36
  • Van Herck H et al. (2001), Blood sampling from the retro-orbital plexus, the saphenous vein and the tail vein in rats: comparative effects on selected behavioural and blood variables. Laboratory Animal Refinement and Enrichment Forum. Animal Technology and Welfare. 4(2) 00-102
  • Dameron GW et al. (1992), Effect of bleeding site in clinical laboratory testing of rats after orbital puncture. 26(1) 53-58
  • Van Herck H et al. (1992) Histological changes in the orbital regions of rats after orbital puncture. 26(1) 53-58
  • Van Herck H et al. (1998), Orbital sinus blood sampling in rats as performed by different technicians: the influence of technique and expertise. Laboratory Animals. 32, 377-386
  • Van Herck H et al. (2000), Orbital sinus blood sampling in rats: effects upon selected behavioural variables. Lab Animal. 34(1) 10-19
  • Timmerman A (1992), Puncture of the tail vein as a possible alternative for orbital puncture in the rat. Animal Technology. 43(3) 167-172
  • Removal of blood from laboratory animals and birds.
  • Toft MF, Petersen MH, Dragstead N, Hansen AK (2005), The impact of different sampling methods on laboratory rats under different types of anaesthesia. Laboratory Animals. 40, 261-274

Retro-orbital sampling in other animals

Click here for information on retro-orbital blood sampling techniques in the mouseClick here for information on retro-orbital blood sampling techniques in the hamster

All blood sampling techniques in the rat

Click here for information on blood vessel cannulation in the ratClick here for information on tail vein blood sampling in the ratClick here for information on jugular vein blood sampling in the ratClick here for information on saphenous vein blood sampling in the ratClick here for information on using a temporary cannula for blood sampling in the ratClick here for information on abdominal/thoracic blood vessel blood sampling in the ratClick here for information on cardiac puncture blood sampling in the ratClick here for information on schedule 1 stunning followed by decapitation for blood sampling in the ratClick here for information on decapitation blood sampling techniques in the rat