- Anesthesia is required for retro-orbital bleeding.
- The following anesthetic agents are recommended for this procedure:
Mice - Ketamine (90 mg/kg) and Xylazine (10 mg/kg), IP
Pentobarbital (50mg/kg), IP
Proparacaine (Ophthetic®) - 1 drop per eye
Isoflurane (Drop Method) - contact vet staff
Rats - Ketamine (75 mg/kg) and Xylazine 10 mg/kg), IP
Pentobarbital (50 mg/kg), IP
- Topical anesthetic, alone, may only be used in mice. Rats, who require more restraint due to their size, must be bled under general anesthesia. Note that there is the potential for contamination of the blood sample if topical anesthetic is used.
- Use of any other anesthetic agents must be identified in the IACUC application.
Prior to and during the procedure the following parameters should be monitored at a minimum of 5 minute intervals:
- Respiratory rate
- Response to noxious stimulus
- Spontaneous movement
- During recovery from anesthesia, the following clinical parameters must be monitored at a minimum of 5 minute intervals until the animal is ambulatory.
· Respiratory rate
· Ability to maintain sternal recumbancy
- To protect the animal from hypothermia they should be placed on a water recirculating heating blanket, or covered well, to conserve body temperature. Animal should never be placed on metal surfaces.
- It is estimated that animals will recover within 30-60 minutes postoperatively.
- Standard heparinized or non-heparinized micro-hematocrit capillary tubes can be used for blood collection.
- The animal is held by the back of the neck and the loose skin of the head is tightened with the thumb and middle finger.
- The tip of the capillary tube is placed at the medial canthus of the eye under the nictitating membrane.
- A short thrust past the eyeball will enter the slightly resistant membrane of the sinus. The eyeball itself remains uninjured.
- As soon as the sinus is punctured, blood enters the tubing by capillary action. It may be helpful to retract the tube to facilitate blood flow.
- When the allowable amount of blood is collected, the tube is withdrawn and slight pressure with a piece of gauze on the eyeball is used to prevent further bleeding.
Analgesia is not required for this procedure but the use of topical anesthetic (e.g., Proparacaine - 1 drop per eye) decreases pain post-procedurally.
- The maximum amount of blood that may be withdrawn at one time from this location is 1% of the animal's body weight (e.g., 0.2 ml from a 20 gm adult mouse and 2.0 ml's from a 200 gm adult rat).
- Blood can only be collected once per week from one eye. Subsequent bleeds should use alternate eyes.
- The maximum number of bleeds for each animal is two bleeds per eye.
- If a project requires a greater volume of blood withdrawal, more frequent bleeds or an increase in the total number of bleeds, any change in the parameters listed must be scientifically justified in the IACUC application.
- Alternate bleeding sites, such as the saphenous or tail veins, should be considered.
Potential adverse effects from this procedure include:
- anesthetic related respiratory distress
- eye infection
- peri-orbital swelling, redness and/or hematoma formation
- Animals should be monitored at least twice weekly after each retro-orbital bleed.
- If adverse effects are seen, the investigator should consult immediately with the veterinary staff regarding treatment options (410-955-3713). If animals have acute adverse reactions to the anesthetic agents (respiratory distress and/or lack of recovery), they must be euthanized immediately.
Animals should be euthanized if the eyeball is acutely damaged, if treatment of an injured/infected eye is unsuccessful and/or if bilateral blindness occurs.