Administering Anesthesia to Mice, Rats, and Hamsters

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Protocol
Administering Anesthesia to Mice, Rats, and Hamsters
Edward A. Greenfield
Anesthesia is used to sedate animals during injections or during operations where unexpected or rapid
movements would endanger the animal. Inhalant anesthetics are often the most appropriate choice to
achieve anesthesia that meets the clinical and humane needs without interfering with the scientific
goals of the research protocol. Isoflurane (i.e., halogenated ether)—the preferred inhalant anesthetic
for small animals—is administered either by placing the animals in a closed container permeated with a
mixture of isoflurane and oxygen vapor, or by using a mask (for larger animals). The depth of
anesthesia is easy to assess and to control. Animals sedated by isoflurane can be handled for injections
or simple operations.
MATERIALS
It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental
Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol.
Reagents
Isoflurane
Indiscriminate use of volatile anesthetics poses safety risks to the user from exposure to waste gases. With
overexposure, isoflurane can cause digestive disorders, irregular heartbeat, and symptoms of drunkenness.
Therefore, isoflurane must only be used in biosafety cabinets, ChemGARD cabinets, and laminar flow
benches, or with waste gas scavenging equipment. It is unacceptable to use isoflurane in open-air situations.
Mice, rats, or hamsters to be immunized
Fractious or difficult-to-control animals pose a heightened risk of bites and/or scratches. In such cases, the use of a
single-animal inhalant system (see Steps 1–5) or an injectable anesthetic is recommended.
Hamsters are a USDA-covered species and will need special record keeping for anesthesia use. Contact your
IACUC and/or staff veterinarian for required training.
Equipment
Anesthesia chamber (for multiple animals)
Equip a Plexiglas box with a removable plastic anesthetic tray and a removable perforated stainless steel floor.
Glass desiccation jars can also serve this function.
Conical tubes, graduated, 50 mL
Modify one of the tubes by punching air holes into the pointed end of the tube.
Fume hood (or other waste gas scavenging system)
Gauze pads or cotton balls
Needle, 1.5 in. × 18-gauge
Syringe, 3-cc
From the Antibodies collection, edited by Edward A. Greenfield.
© 2019 Cold Spring Harbor Laboratory Press
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot100198
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METHOD
This protocol is adapted from the Dana Farber Cancer Institute.
Anesthetizing a Single Mouse or Rat
1. Place a few gauze pads or cotton balls inside an unmodified 50-mL graduated conical tube. Push
the pads to the bottom of the tube.
2. Using the needle and syringe, draw up 1 mL of isoflurane, and transfer it onto the gauze pad in the
bottom of the tube. Immediately screw on the tube cap.
Dispose of the needle and syringe in an appropriate sharps container.
3. Insert the conical tube modified with holes cut into the tip into the first tube.
This second tube will hold the animal to be anesthetized and prevent the animal having any direct contact
with the isoflurane.
4. Place a mouse or rat into the second tube (Fig. 1), and anesthetize the animal.
Never place an animal in direct contact with the isoflurane-soaked gauze pad.
5. When the rodent is anesthetized (see Discussion), remove it from the tube. Immediately separate
the tubes. Recap the tube containing the isoflurane.
The isoflurane tube can be used again, often without adding additional isoflurane. Add additional isoflurane
only when necessary. Outer tubes can be changed and decontaminated between animals to avoid spread-
ing any opportunistic pathogens.
Anesthetizing Multiple Animals
6. Place a few gauze pads or folded paper towels in the removable plastic tray of the anesthesia
chamber. Place the tray inside the box.
7. Pour 2–4 mL of isoflurane onto the gauze pads. Put the stainless steel floor in place. Immediately
close the chamber to prevent evaporation of the anesthetic.
Alternatively, use a machine to deliver the anesthetic into the chamber (Fig. 2).
The chamber is now ready to receive animals.
8. Place 1–5 mice or 1–2 rats into the chamber.
The actual number of animals placed in the chamber will depend on the size of the animals and the
experience of the individual performing the anesthesia.
FIGURE 1. Proper method of anesthetizing a mouse with isoflurane.
458 Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot100198
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 Cold Spring Harbor Laboratory Press
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9. Carefully monitor all of the animals in the chamber to ensure that they do not overdose. Leave the
animals in the chamber until the first and last animal in the chamber are completely anesthetized
but not overdosed (see Discussion).
Limiting the number of times and duration the chamber lid is opened is imperative to reducing exposure.
Experienced personnel should perform this procedure because improper technique can lead to excessive
exposure to isoflurane vapors.
DISCUSSION
Assessing Depth of Anesthesia
A mouse or rat is fully anesthetized when it has a:
• shallow, slow, rhythmic breathing pattern. (An alert rodent has a rapid breathing pattern.)
• lack of pedal reflex. To judge the depth of anesthesia, pull the animal’s hind leg out straight and
pinch the foot hard using your thumb and forefinger. Animals should not be operated on until the
withdrawal reflex has abated.
• lack of palpebral (blink) reflex, which can be determined by lightly touching the eyelid with a
gauze pad.
Postprocedure Monitoring
For procedures in which an animal is anesthetized for longer than 5 min, use eye ointment to prevent
corneal drying from the lack of a blink reflex.
Monitor all animals until they have recovered from anesthesia. After returning the animal to its
cage, check frequently until the animal is able tomaintain sternal recumbence and is able to reach food
and water.
FIGURE 2. Anesthesia machine for use with mice and rats. (A) Anesthesia apparatus. (B) Inhalation chamber. (C )
Anesthesia flow regulator.
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot100198 459
Administering Anesthesia to Mice, Rats, and Hamsters
 Cold Spring Harbor Laboratory Press
 at NYU MED CTR LIBRARY on July 21, 2019 - Published by http://cshprotocols.cshlp.org/Downloaded from 
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doi: 10.1101/pdb.prot100198Cold Spring Harb Protoc; 
 
Edward A. Greenfield
 
Administering Anesthesia to Mice, Rats, and Hamsters
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