- Cotton pad (ie. nestlet or gauze)
- Bell jar or other small container with a secure lid (with known volume)
- Wire mesh to fit the bottom of container
- Isoflurane mixture- 20% v/v isoflurane in propylene glycol for mice
- Isoflurane mixture- 30% v/v isoflurane in propylene glycol for rats
- OEHS-approved hood
For brief procedures:
- Under a hood and wearing gloves, wet the cotton pad with the isoflurane mixture (use ~1.0 cc of mixture for every 500 cc volume of jar) and place gauze in the jar underneath the wire mesh to prevent direct contact of animal with the isoflurane soaked pad. Alternatively, place a conical tube containing an isoflurane soaked gauze into the container (see picture below). (Note- undiluted isoflurane is not recommended because it can lead to lethal accumulations of anesthetic in the vapor phase).
- Place animal in anesthesia jar and close lid tightly. Monitor animal closely. A deep plane of anesthesia is indicated by lack of a righting reflex when the jar is tipped slightly and a respiratory rate that is reduced by ~50% from pre-anesthetic rate (i.e. 80-100 breaths/min). This should take ~1 min for mice and ~2 min for rats.
- Allow animal to remain in deep anesthesia for ~10 sec before proceeding. Remove animal from the jar and replace lid immediately.
- To ensure an adequate plane of anesthesia, apply a noxious stimulus (i.e. toe pinch) before proceeding with the procedure. If the animal responds to the toe pinch return it to the jar.
For prolonged procedures:
To maintain anesthesia for a longer period of time (up to ~ 8 min), a nose cone can be constructed using a 15 ml conical tube for mice and a 50 ml conical tube for rats. To make the nose cone, place a small piece of gauze moistened with the isoflurane mixture inside the tube pushed towards the end of the tube so that it will not come in direct contact with the animal.
Anesthetize the animal in the isoflurane container as described above. When the animal has reached the desired depth of anesthesia, remove it from the jar and place the animal’s muzzle at the edge of the nose cone taking care not to create a complete seal. The depth of anesthesia can be adjusted by moving the nostrils closer or farther away from the end of the cone.
References: Itah et al., 2004. A replacement for methoxyflurane (Metofane) in open-circuit anesthesia. Lab. Anim. 38:280-5.
STEP 1: Place animal in bell jar or other small container with a conical tube containing the isoflurane soaked gauze or in a container with a wire mesh bottom with the isoflurane soaked gauze underneath the mesh so that the animal does not come into direct contact with the isoflurane mixture.
Remove the animal when it has reached the desired depth of anesthesia.
For longer procedures: Complete Step 1 then proceed as follows:
STEP 2: Construct a nose cone using a conical tube (15 ml for mice, 50 ml for rats) with a pad soaked in the isoflurane mixture pushed to the end of the tube. Once the animal has reached the desired depth of anesthesia (Step 1) remove from the container and place the nose cone at the end of the animal’s muzzle being careful not to create a vacuum. The depth of anesthesia can be controlled by moving the nose cone closer or farther away from the nostrils (see below).