Anesthesia Guidelines: Mice

Find guidelines for mouse anesthesia, including anesthetic drugs and procedures and special considerations for neonatal mouse anesthesia.

Research Animal Resources (RAR) veterinary staff designed these guidelines to be general recommendations, not an inclusive list of all possible drug combinations that can be used in mice. Consequently, guidelines do not reference specific research-associated concerns.

If you have questions about using anesthetics for your particular situation, please work with your area veterinarian to develop the most effective anesthetic plan.


How to use anesthesia

Animals may be anesthetized for surgery, non-surgical procedures that may be painful, or non-painful procedures that require immobilization. Anesthesia causes the loss of feeling in all or part of the body, with or without loss of consciousness.

Steps must be taken before, during, and after anesthesia to ensure animal safety and anesthesia efficacy.  

Anesthetic drugs can be administered parenterally or by inhalation. The choice of anesthetic agent will depend on the procedure to be performed, research aims, and other factors such as animal age; there are special considerations for neonatal mice. Consult your area veterinarian with questions about drug selection.


General guidelines and considerations for mouse anesthesia

Acclimation

A one-week acclimation period is recommended for all animals to prevent stress-induced disease.

Fasting

Pre-anesthetic fasting is usually not necessary for mice due to their inability to vomit. However, if fasting is employed, limit it to no more than two to three hours due to the high metabolic rate of small rodents. Never restrict water.

Eye protection

Mouse eyes remain open under anesthesia. This can lead to corneal drying and trauma. Apply ophthalmic ointment (e.g., Paralube® or Lacrilube®) to eyes if:

  • The anesthetic event lasts longer than five minutes.
  • Anesthesia is being delivered by facemask. 

Monitoring

Regardless of the anesthetic administered, monitor mice under anesthesia to avoid excessive depression of cardiac and respiratory functions, or insufficient anesthesia. Parameters that can be monitored in an anesthetized mouse without specialized equipment include:

  • Anesthetic depth: Toe pinch. 
  • Respiratory rate and pattern: The normal undisturbed rate is ~180/minute, a slow rate drop of 50% is acceptable during anesthesia.
  • Mucous membrane color: Should be pink, not blue or gray.
  • Body temperature: Can be monitored with a rectal thermometer, and should be above 97℉.
  • Oxygen saturation and heart rate: Can be monitored using a specialized rodent pulse oximeter, and should be greater than 95% and between 300-800 beats/minute, respectively. 

Heat support

All species are at risk for hypothermia while under anesthesia. Mice are particularly susceptible due to their high body surface area to body mass ratio. Hypothermia induces significant physiological stress on animals that can prolong recovery and potentially be fatal. Guidelines:   

  • Provide supplemental heat during all anesthetic events.  Supplemental heat sources include circulating water blankets, electric heating pads, and commercial products that can be warmed in a microwave.  
  • Never place the animal directly on the heat source.  

Fluid support

Consider providing warm subcutaneous (SQ) or intraperitoneal (IP) fluids, particularly for prolonged anesthetic events or animals that are ill, aged, or debilitated. 

Recovery

Continue to monitor animals until they are fully recovered. Guidelines:

  • Recover animals on paper towels (without bedding) in a clean cage. This minimizes the risk of tracheal obstruction or pneumonia.
  • Recover anesthetized animals alone in a cage.
  • Continue to provide supplemental heat during recovery.
  • When the animal is ambulatory, return it to the home cage with immediate access to food and water.

Contact your area veterinarian with questions or for additional information. 


Anesthetic drugs and procedures

Inhaled anesthetics

Inhaled anesthetics


Isoflurane – recommended

Dose: 4-5% for induction; 1-2% for maintenance
Comments: 300μL in a 500ml container for drop jar anesthesia

Injectable anesthetics

Injectable anesthetics

Combined ketamine/xylazine is the preferred injectable anesthetic in mice. Other commonly used injectable agents are listed below.

Key considerations:

  • Mice vary significantly in their sensitivity to various anesthetics. Age, body composition, strain, health status, genetic manipulation, and sex are just a few factors that can contribute to anesthetic sensitivity. The following doses are general guidelines that may vary significantly based on the aforementioned factors. When adding anesthetics to your protocol, it is advisable to provide a range to allow titration for the specific needs of each animal.
  • Intramuscular (IM) injections are not recommended in mice as it can result in complications such as tissue irritation, lameness, and self-mutilation.

Ketamine + Xylazine (Rompun®) – recommended

Dose: Ketamine: 80-120 mg/kg; Xylazine: 5-10 mg/kg
Route: Intraperitoneal (IP)
Duration: 30-45 minutes
Comments: Thermal support is crucial. If additional anesthetic is needed, supplement with 1/3 dose of ketamine only. Do not re-dose xylazine. Xylazine can be reversed with atipamizole (Antisedan®, 1-2 mg/kg SQ or IP), or yohimbine (Yobine®, 0.5-1 mg/kg IP).


Ketamine + diazepam (Valium®)

Dose: Ketamine: 100 mg/kg; Diazepam: 5 mg/kg
Route: IP
Duration: 20-30 minutes


Pentobarbital (Nembutal®)

Dose: 30-40 mg/kg (sedation); 40-60 mg/kg (anesthesia)
Route: IP
Duration: 10-300 minutes
Comments: Causes respiratory depression; narrow margin of safety. Use with caution.


Tribromoethanol (Avertin®) – not recommended

Dose: 225-240 mg/kg
Route: IP
Duration: 30-45 minutes
Comments: Refer to the IACUC guideline on using Avertin® (2,2,2 Tribromoethanol) for use specifications.


Alpha-chloralose – not recommended

Dose: 115 mg/kg
Route: IP
Duration: Unproven


Chloral hydrate – not recommended

Dose: 400 mg/kg
Route: IP
Duration: Unproven

Local anesthetics

Local anesthetics

Local anesthetics are a type of injectable anesthetic. Local anesthetics block nerve impulses by specifically binding the voltage‐gated Na+ channel in the nerve cell membrane.

Routes of administration include topical to mucous membranes (nose, eye, etc.), or an injection directly into tissues and around nerve bundles.

Key considerations:

  • Administration of local anesthetics prior to the painful stimulus (e.g., incision) would be considered an adjunct analgesic to opioid and NSAID analgesics. 
  • Use as a primary analgesic is discouraged due to the short duration (hours).
  • Lidocaine has a quick onset (1-2 minutes), but is short-acting (1-2 hours). Bupivacaine has a slow onset (10-15 minutes), but is long-acting (4-8 hours). When used in combination (lidocaine plus bupivacaine in the same syringe), local anesthesia can have a fast onset with a relatively long duration of action.
  • For rodent use, dilute 1‐2% lidocaine to 0.5% and 0.5% bupivacaine to 0.25% to allow for feasible volumes to infuse at the incision site (1% solution is equal to 10 mg/mL).

Lidocaine

Dose: 4 mg/kg (0.4 ml/kg of a 1% solution)
Route: Local infiltration
Comments: Do not exceed 7 mg/kg total dose. Rapid onset, short duration.


Bupivacaine

Dose: 1-2 mg/kg (0.4-0.8 ml/kg of a 0.25% solution)
Route: Local infiltration
Comments: Do not exceed 6 mg/kg total dose. Slower onset, longer duration.


Special considerations: Neonatal mouse anesthesia

The literature currently describe several anesthetic methods for use in neonatal rodents, including injectable anesthesia, inhalant anesthesia, and hypothermia.

Parental cannibalism is a problem with neonatal rodent anesthesia. This can be reduced by ensuring the neonate is fully recovered before returning it to the dam. Additional steps include exposing pups to soiled bedding from the mother’s cage, and placing the pup in the middle of the litter.

Hypothermia anesthesia

Hypothermia anesthesia

  • Hypothermia should only be performed in neonatal rodents <7 days old and should not be used for procedures lasting longer than 30 minutes.
  • Animals should not be placed directly on ice (use latex glove or another substance between the animal and the underlying ice bath).
  • Animals have reached the proper plane of anesthesia when pedal reflex is lost.
  • Do not use incandescent light during the procedure, as it can warm the surgical field and cause animals to awaken from a surgical plane of anesthesia.  
  • Following anesthesia, the animal should be re-warmed slowly. Rapid warming can cause tissue damage. Patients can be re-warmed on a circulating water blanket, heating pad (40°C), or in an incubator (33°C).
  • Pups can be returned to dam once they are able to move without direct physical stimulation.
  • Refer to the IACUC guidelines on using hypothermia as anesthesia for neonatal rodents

Inhalation anesthesia

Inhalation anesthesia

Neonatal rodents may have a longer induction time than adult rodents with inhalant anesthesia. Induction in a chamber generally requires 5% isoflurane, and maintenance (chamber or nose cone) requires 1-2% (flow rate = 0.5-1 L/min).

Injectable anesthesia

Injectable anesthesia

Ketamine/xylazine is the recommended injectable anesthetic for mice >7 days of age. Inject 50-150 mg/kg ketamine and 5-10 mg/kg xylazine with a 27G needle IP (max volume = 0.5 ml).


References for these guidelines are available by request.