Open-Drop or Nose Cone Method of Isoflurane Anesthesi= a in Mice and Rats

Division of Laboratory Animal Resources (DLAR)

University of Kentucky Medical C= enter

PURPOSE<= /u>: This procedure details how to use an isoflurane/propylene glycol mixture to anesthetize rodents. Isoflura= ne is an inhalant anesthetic designed for use with a precision vaporizer. This me= thod should be reserved for instances where short duration anesthesia is desired= or it is impractical to use a precision vaporizer.

The open-drop method of isoflurane exposure can be used to anesthetize mice and rats for brief non-surgical procedures. To maintain anesthesia for 5-10 min= utes duration, a simple nose cone can be constructed from a syringe with the plu= nger removed.

Note that even when diluted, this method does not allow for more than crude cont= rol of the concentration of the anesthetic and therefore the potential for inadvertent overdose with this method is significantly greater than when a precision vaporizer is used. = Mice and rats need to be watched very closely and observed during induction to a= void anesthetic overdose and death.

APPLICATION: The open-drop = and nose cone method of inducing isoflurane anesthesia is an alternative to the= use of a precision vaporizer where short duration anesthesia is desired for min= or procedures in rodents. Precis= ion vaporizers provide a much safer and more reliable method of inducing isoflu= rane anesthesia. The open-drop and= nose cone procedures are not acceptable for major surgical procedures or procedu= res requiring greater than 10 minutes of surgical anesthesia or where the use o= f a precision vaporizer is required to maintain an adequate and safe level of anesthesia.

Important Safety Consideration: Because of risks to human health, the = use of inhalant anesthetic is not recommended on open bench tops. The following procedures should only be performed in a certified ducted hood, biosafety cabinet or portable ductless hood equipped with a charcoal filter. The Department of Environmental Health and Safety should be consulted regarding appropriate scavenging methods to mitigate human exposure to excess anesthe= tic agent.

PROCEDURES:=

Materials:

·      =    Cotton pad = (i.e.cotton ball, nestlet = or gauze pad)

·      =    Container (= with known volume)*

·      =    Wire mesh to fit in bottom of container (using a histology tissue cassette to contain the gauze might be a helpful alternative option to the wire mesh.)<= o:p>

·      =    Mixture of = 20%v/v isoflurane** in propylene glycol (for mice)

·      =    Mixture of = 30% v/v isoflurane** in propylene glycol (for rats)

·         Certified ducted hood, biosafety cabinet or portable ductless hood equipped with a charcoal filter. <= /p>

Optional Materials:<= o:p>

·         To maintain anesthesia for up to 10 minutes duration, a simple nose cone can be constru= cted from a 3cc syringe (for mice; larger syringe for rats) with the plunger removed or a conic= al tube (15ml for mice; 50 ml for rats). A pre-cut section of gauze is also required for the nose cone.

Any type of container with a secure lid may be used, provided it is constructed= of non-porous material that is sanitizable and allows for constant visualizati= on of the animal. Ensure that the animal does not contact the isoflurane-soaked pad, which can cause skin irritation and potential overdosing since isoflur= ane is also absorbed through skin. The container should be of sufficient size to comfortably accommodate the anima= l, but not so large as to require excessive anesthetic.

** Use of isoflurane without propylene glycol dilution is unacceptable, as the vapor pressure of isoflurane will lead to lethal accumulations of anesthetic in the vapor phase.** =

Open Drop Procedu= re (For brief procedures)= : Mice will remain deeply anesthetized f= or approximately 30 seconds and rats for one minute. This method can be used f= or retro-orbital blood sampling, tail biopsies and similar rapid procedures. To maintain longer anesthetic times, see Nose Cone Procedure. <= /o:p>

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1.    <= /span>Don gloves. Open bottle containing appropriate isoflurane/propylene glycol mixture in approved hood. Wet a cotton pad (e.g= ., nestlet, gauze) with an exact amount of isoflurane mixture. A 1.0 cc volume= of appropriate mixture should be used for every 500 cc volume of the anesthesia container.

2.    <= /span>Place the cotton pad inside a small container un= der a wire mesh. The use of the mesh ensures that the animal does not contact the isoflurane-soaked pad, which can cause skin irritation and potential overdo= sing since isoflurane is also absorbed through skin.

3.    <= /span>Place = animal in anesthesia jar and close lid tightly. Monitor animal closely. A de= ep 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. O= bserve the animal closely so that overdose does not occur.

4.    <= /span>Allow = animal to remain in deep anesthesia for ~10 sec before proceeding. Remove an= imal from the anesthesia container and replace lid immediately.

5.    <= /span>To ens= ure an adequate plane of anesthesia, apply a noxious stimulus (i.e. toe pinch) bef= ore proceeding with the procedure. If the animal responds to the toe pinch return it to the anesthesia container.

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Nose Cone Procedure This method = can be used with the Open Drop Procedure above to maintain anesthesia for up to 10= minutes duration and is appropriate for minor surgical procedures, such as subcutaneous tum= or implantation. Procedures lasting longer than 10 minutes should be performed using a precision vaporizer system.

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<= span style=3D'mso-list:Ignore'>1.&n= bsp;   Slightly moisten the end of a small piece of gauze with the Isoflurane mixture. Inse= rt the gauze into syringe nose cone of appropriate size, with moistened end aw= ay from open end of the nose cone. Place prepared nose cone into anesthesia ja= r, until ready to use.

<= span style=3D'mso-list:Ignore'>2.&n= bsp;   Anesthetize the animal as described in steps #1-4 (Open Drop Procedure) above.

<= span style=3D'mso-list:Ignore'>3.&n= bsp;   Retrieve the nose cone from the jar, and still working in the hood, place the animal’s muzzle at the edge of the nose cone. Moisten the animal̵= 7;s eyes with Paralube. Check the depth of anesthesia as described as step #5 a= bove (the depth of anesthesia can be adjusted by moving the animal’s nostr= ils closer to or further from the end of the cone). Care must be taken not to create a complete seal around the muzzle.

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        &= nbsp;   Fig.1 Take care not to push the = cone completely        &= nbsp;           &nbs= p;    Fig.2 Watch animal closely for signs of

        =    against the muzzle. Allow some space on e= ither side          awakening and reposition conical= tube as in Fig.1        &= nbsp;         &= nbsp;

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<= span style=3D'mso-list:Ignore'>4.&n= bsp;   If appropriate, perform the procedu= re.

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<= span style=3D'mso-list:Ignore'>5.&n= bsp;   Allow the animal to recover on a piece of clean paper towel in a bedding-free recovery cage to prevent aspiration injury or death. Monitor the animal clo= sely until it can maintain sternal recumbency, then transfer to the home cage. <= o:p>

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6.    Air dry isoflurane exposed cott= on pad(s) and nose cones in the hood for 15 minutes and discard by wrapping in= a glove and transferring to a biomedical waste disposal bag or bucket. <= /o:p>

Important Safety Consideration: Because of risks to human health, the = use of inhalant anesthetics is not recommended on open bench tops. The above procedures should only be performed in a certified ducted hood, biosafety cabinet or portable ductless hood equipped with a charcoal filter. The Department of Environmental Health and Safety should be consulted regarding appropriate scavenging methods to mitigate human exposure to excess anesthe= tic agent.

**As a reminder DLAR does not have ducted hoods or charcoal filtered hoods in any animal housing room. This method c= annot be used in animal housing rooms. Investigators should contact DLAR for locations of appropriately duc= ted hoods where this can be safely performed in the vivarium procedure rooms.**

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For qu= estions or training, please contact any DLAR Veterinarian or the DLAR Training Coor= dinator.

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REFERENCES: <= /u>

<= span style=3D'mso-list:Ignore'>1.&n= bsp;   Itah et al. 2004. A replacement for methoxyflurane (Metofane) in open-circuit anesthesia. Lab.Anim. 38:280-5.

<= span style=3D'mso-list:Ignore'>2.&n= bsp;   Nagate, T., T. Chino, et al. (2007). "Diluted isoflurane as a suitable alterna= tive for diethyl ether for rat anaesthesia in regular toxicology studies." Journal of Veterinary Medical Science 6= 9(11): 1137-1143.