Download Rodent Anesthesia Techniques

Rodent Anesthesia techniques
Guidelines: The procedures described in this SOP should be followed to anesthetize rodents for
survival or terminal procedures. Inhalant anesthesia using the rodent EZ anesthesia machine
located in B16G of the ARF surgery suite is recommended for most survival procedures due to
its safety and the ease of controlling the depth and duration of anesthesia. Injectable anesthesia is
recommended for terminal procedures, or when rodents cannot be transported to the B16 surgery
suite, i.e. immunocompromised animals, BSL-2 projects, etc.
Materials:
Rodent EZ anesthesia machine
Isoflurane
Ketamine- 100mg/mL stock
Xylazine- 100mg/mL stock
Telazol®- 500mg lyophilized vial
Acepromazine- 10mg/mL
Diazepam- 10mg/mL stock
Sodium pentobarbital (Nembutal®)- 50mg/mL stock
Sterile water for injection
Sterile needles- 25G, 27G
Sterile syringes-1CC
Gauze
Rodent restrainer
Gram scale
For accurate calculation of injectable drug dosages animals should be weighed. When working
with a group of animals of the same age, strain, and gender, the average weight can be used for
calculating dosages.
Inhalant anesthesia using the rodent EZ anesthesia machine
The rodent EZ anesthesia machine is located in B16G. Anesthesia machine use must be
scheduled in advance using the Anesthesia Machine Groupwise calendar. Contact Dr. Kathryn
Jones for access to the Groupwise calendar.
1. Turn on the heating unit for the operating stage and allow it to warm for at least 15 minutes
prior to performing procedures.
2. Wearing gloves and a mask, fill the vaporizer with isoflurane.
a. Make sure the oxygen regulator and vaporizer are turned off completely before
opening the injection port.
b. Remove the cap from the injection port on the Isoflurane vaporizer.
c. Carefully pour in enough isoflurane to have the meniscus between the two guide lines
on the fill window. Using a syringe and needle to fill the vaporizer will minimize
spills.
d. Replace the cap on the injection point and finger tighten until it is secure.
3. Release the latch on the front of the induction chamber and lift the upper chamber.
4. Place animals in the lower chamber by gently pushing them through the overlapping seam on
the silicone membrane. Multiple animals can be placed in the lower chamber
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Rodent Anesthesia techniques
5.
6.
7.
8.
Close and latch the upper chamber.
Turn on the laminar flow system using the toggle switch on the cord.
Turn on the oxygen flowmeter on the vaporizer by turning the knob counter clockwise
Turn on the oxygen tank to no more than 2L/minute. Adjust the flowmeter on the vaporizer
to 1L/minute for mice or 1.5L/min for rats and hamsters.
9. Turn on the dial on the top of the isoflurane vaporizer to 5% to start the flow of anesthetic
into the lower chamber.
10. Observe the animals until they stop moving and their breathing slows, usually 2-5 minutes.
11. Reduce the concentration of isoflurane by turning the vaporizer dial down to 1.5-2.5%.
12. Turn the oxygen flowmeter on the vaporizer down to 0.5L/min. This will increase the flow of
anesthetic to the breather unit for the surgical stage while maintaining an adequate level in
the induction chamber.
13. To remove an animal from the induction chamber, reach through the hand port on the upper
chamber and through the silicone membrane into the lower chamber. Quickly remove the
animal and place it on the surgical stage.
14. Remove the plug from the nose cone and place the animals’ nose in the nose cone, being sure
that the nostrils are not pushed too far into the cone to occlude breathing.
15. To test the depth of anesthesia, firmly pinch one paw between the fingers. If there is no
purposeful movement or vocalization, the animal is anesthetized enough to proceed. If there
is purposeful movement or vocalization, allow the animal to inhale the isoflurane for 1-2
minutes more before retesting the animal.
16. Once the procedure is complete, remove the animal from the surgical stage and place it in a
cage either on its side or sternum. Observe the animal continuously until it is able to stand
and walk normally. This usually occurs 5-10 minutes after it is removed from the isoflurane.
17. Once all procedures are done and the animals have been returned to their cages, replace the
stopper in the nose cone and shut down the EZ anesthesia system as follows:
a. Turn off the oxygen tank and close the oxygen flowmeter by turning the dial
clockwise.
b. Turn of the vaporizer by turning the dial to 0.
c. Pull open the flush valve, located in the lower right corner of the unit, and secure it
with the latch.
d. Wait 2 minutes to allow all isoflurane to be scavenged from the unit.
e. After the isoflurane has been scavenged, release the latch to close the flush valve.
f. Turn off the laminar flow system using the cord toggle switch.
18. Clean the induction chamber after the procedure is complete as follows:
a. Release the latch on the front of the induction chamber and lift the upper chamber.
b. Lift the top of the lower induction chamber out of the chamber.
c. Clean all debris out of the lower chamber using paper towels and MB-10 disinfectant.
d. Replace the top of the lower induction chamber, then close and secure the latch on the
upper induction chamber.
e. Wipe down the surgical stage with MB-10 disinfectant.
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Rodent Anesthesia techniques
Injectable anesthetic combinations
Below are some recommended anesthetic drug combinations that generally provide 30-60
minutes of anesthesia, depending on age, sex, and strain of animal. Doses at the higher end of the
range are used to ensure a surgical plane of anesthesia, which is suitable for terminal procedures.
All drugs are given IP, except where indicated.
Drug combination
Pentobarbital
Ketamine +Xylazine
Ketamine
+Acepromazine
Ketamine +
Diazepam
Telazol+ Xylazine
Mice
50-90mg/kg
100mg/kg (K) +
10mg/kg (X)
100mg/kg (K) + 25mg/kg (A)
100mg/kg (K) +
5mg/kg (D)
80mg/kg (T) +
10mg/kg (X)
Dosage
Rats
30-45mg/kg
75-95mg/kg (K) +
5mg/kg (X)
75mg/kg (K) +
2.5mg/kg (A)
75mg/kg (K) +
5mg/kg (D)
40mg/kg Telazol
only
Hamsters
50-90mg/kg
80mg/kg (K) +5mg/kg
(X)
100mg/kg (K) IP + 14mg/kg (A) IM
40-100mg/kg (K) +
5mg/kg (D)
30mg/kg (T) +
10mg/kg (X)
1. Weigh the animal or group of animals and calculate the appropriate dose of the selected
anesthetic.
2. Prepare the syringe with the anesthetic and remove all air bubbles.
3. Administer the injection following the rodent injection SOP.
4. Place the animal in its cage and observe until the anesthesia has taken effect, generally 5-10
minutes.
5. To test the depth of anesthesia, firmly pinch one paw between the fingers. If there is no
purposeful movement or vocalization, the animal is anesthetized enough to proceed. If there
is purposeful movement or vocalization, wait an additional 2-3 minutes more before retesting
the animal.
6. For survival procedures:
a. Apply a small amount of sterile eye lubricant to both eyes to protect the corneas.
b. Once the procedure is complete, place the animal in its cage either on its side or
sternum. Observe the animal continuously until it is able to stand and walk normally.
Turn the animal every 10-15 minutes to the other side to prevent pressure injuries on
one side.
c. Once the animal is fully recovered, it can be returned to the ARF holding room.
7. For terminal procedures:
a. Once the procedure is complete, euthanize the animal either by cervical dislocation or
thoracotomy.
b. Place the carcass in a biohazard bag and store in an ARF carcass disposal freezer.
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Rodent Anesthesia techniques
Post-procedural monitoring:
1. Anesthetized animals must be monitored continuously until they are awake and able to walk
normally.
2. Awake animals should be monitored at a minimum 1 hour and 24 hours after the procedure
to ensure full recovery.
3. Observe the animals for the following signs:
a. Bleeding from any incisions, blood collection, or injection site
b. Labored breathing
c. Discharge from the nose or mouth
d. Ruffled fur
e. Hunched posture
f. Lethargy
g. Minimal responsiveness
4. Lethargy, labored breathing, minimal responsiveness and uncontrolled bleeding or discharge
are criteria for immediate euthanasia.
5. Document all observations on the experimental health monitoring sheet. Alert the Attending
Veterinarian of any ill or injured animals.
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