Retrobulbar Injection & Retro-Orbital Bleeds
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Retrobulbar Injection & Retro-Orbital Bleeds
University of South Florida
Division of Comparative Medicine
July 2014
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Retro-Orbital Sinus Injection
• Is an injection into the ophthalmic venous sinus (mice) or plexus (rats), or a retrobulbar injection
• Is suited for the injection of non-irritating substances into darkly pigmented mice, hamsters, or mice and rats with no discernible tail veins available for injection
• Distribution of substances injected retro-orbitally is similar to that of the intravenous route with high blood levels achieved rapidly
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Retro-Orbital Injection (ROI)
• Retro-orbital injection allows multiple injections (must alternate eyes) and causes minimal distress if done correctly
• This procedure is to be done only on approved protocols listing this route or in extreme circumstances with veterinary staff approval
• Anesthesia is always required for injection; Isoflurane is recommended to speed recovery.
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RO Injection
• Injection volumes should not exceed 0.1 to 0.3 ml in mice
• no more than two injections (one per eye) should be performed on one animal, unless the protocol states otherwise
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ROInjection
• A 28 to 25g needle for mice (28-23g in rats) with loaded syringe attached is introduced retro-orbitally via the medial canthus
• As one advances the needle into position, the bone beneath can be felt and used as a guide
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RO Injection
• The bevel should be positioned towards the bone of the orbit.
• Once placement has been achieved, indicated by a slight “bump” as the tip reaches the slight depression felt where the ophthalmic veins come together
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RO Injection
• The plunger may be depressed and the material delivered
• Slight downward pressure will result in the veins being punctured for an intravenous injection
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RO Injection
• Too much pressure will result in the arteries being ruptured or the bones fractured and the compound may be lost in the resulting hemorrhaging
• The technician will need to apply lubricant to the eye following injection to prevent the cornea from drying/injury
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RO Injection
• Proper placement and injection technique will result in a visible ‘bulging’ or blanching of the eye with no leakage of material
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RO Injection
• Upon delivery, the needle may simply be backed out and the eye will return to its normal position.
• If the eye ruptures, appears damaged or dried out following injection(s) then veterinary care must be sought and the animal treated or euthanized as directed.
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Retro-Orbital Sinus Blood Collection in Mice
• The retro-orbital site for blood sampling has been associated with a high potential for inducing orbital and ocular pathology, therefore other routes should always be considered.– increased observation frequency after retro-orbital
sampling should occur.• The method has been associated with greater
incidence of sample hemolysis and increased levels of corticosterone and glucose.
• http://www.medipoint.com/html/mouse_phlebotomy.html
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Retro-Orbital Bleeds• Signs that would indicate problems
associated with ocular pathology:– swelling or injury of the eye, hematoma
behind the globe, opacity of the lens, overt guarding of the eye, corneal opacity or ulceration, excessive tearing, expathathalomos (bulging), self trauma, conjunctivitis.
· If complications manifest, animals will be treated/euthanatized in accordance with the attending veterinarian’s recommendations.
Corneal opacity
exophathalmos
Ulceration/tearing
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Retro-Orbital Bleeds
(ROB)
• The retro-orbital sinus (mouse) and plexus (rat) is a system of dilated venous channels at the back of the orbit.
• Blood can be collected from this area in anesthetized animals using a sterile hematocrit tube.
• Anesthesia is always required; Isoflurane is recommended.
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RO Bleedso Once anesthetized, the animal is
placed on a flat surface, and the handler’s thumb is used to apply pressure to the external jugular vein just behind the lower jaw.
o The index finger of the same hand is used to pull the upper eyelid back and help ‘pop’ the eye up.
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RO Bleeds• The other hand is then used to introduce a sterile hematocrit capillary tube along the inner corner of the eye (medial canthus) beside the eyeball.
• There should be no movement of the head during the procedure.
• Damage to the optic nerve and other intra-orbital structures, which can lead to deficits in vision and even blindness
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RO Bleeds• Pressure is exerted from the thumb and forefinger just behind the eye and the skin pulled back to allow the eyeball to protrude.
• The tube is positioned , and gently inserted through the conjunctiva towards the back of the eye sliding along the orbit.
• Fracture of the fragile bones of the orbit and neural damage by the micropipette; and penetration of the
eye globe itself with a loss of vitreous humour.
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RO Bleeds
• Rotation of the tube gently while advancing assists positioning, penetration of conjunctiva and rupturing of the sinus and allows blood to flow freely when properly inserted.
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RO Bleeds
• Tilting the head slightly downward may improve flow.
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RO Bleeds
• Bleeding will stop when the hematocrit tube is withdrawn, pressure to the jugular is released and the eyeball is allowed to fall back into its normal position.
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RO Bleeds
• Light pressure applied to the closed eyelids will assist with hemostasis.
• Gently shut the eye as the tube is removed by squeezing the lids together.
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RO Bleeds
• Immediate indication of damage as evidenced by the eyeball not returning to a normal position following the bleed or observance of nasal bleeding should be brought instantly to the attention of the veterinary staff.
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RO Bleeds• No more than 10% of
the blood volume should be removed at one sampling
• The blood volume of a mouse is approximately 8% of the body weight.
• Example:
25g mouse = ~2ml bld
volume = bleed of ≤
200μl max
Each micro hematocrit tube holds ~ 75μl
Use heparinized capillary tubes to prevent clotting during collection
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RO Bleeds• This procedure may be done repeatedly
– should not be done in excess of six times per animal
– no more than three alternating bleeds per eye• Collection volume of 10ml/kg, at an interval of
once every 2 weeks will not be exceeded unless otherwise approved
• Additional information can be found at:– http://oacu.od.nih.gov/ARAC/documents/Rodent_Bleeding.pdf – http://www.theodora.com/rodent_laboratory/blood_collection.html