Guidelines on Anesthesia and Analgesia in Mice

Unit for Laboratory Animal Medicine
May 19, 2023 12:00 am

This document has been designed by the ULAM veterinary personnel as a guideline for sedation, anesthesia, and analgesia of laboratory mice. This is not intended to be an inclusive tutorial on all possible drug combinations that can be used in mice. The following guidelines are also general recommendations and consequently do not include reference to specific research associated concerns. 

All surgical procedures, anesthetics, analgesics, antibiotics or other medications used on animals must be approved by the IACUC, described in the animal use protocol and performed by personnel listed on the protocol and appropriately trained for the surgical procedure. Any techniques or drug protocols deviating from this document must be justified and approved in the IACUC protocol prior to application.

  • Responsibility

    1. Principal Investigator: Responsible to ensure appropriate anesthesia and/or analgesia is provided for all rodents undergoing painful procedures including rodent survival surgery unless otherwise indicated in the relevant approved protocol.
  • Glossary Definitions

    Acclimation Period

    The time period provided to an animal after shipment to allow physiological and psychological stabilization prior to any experimental manipulation.


    This encompasses both of the following definitions:

    1. Local Anesthesia: Temporarily induces loss of sensation to a specific part of the body. May provide pain relief.
    2. Systemic Anesthesia: Temporarily induces loss of sensation with loss of consciousness. Only provides pain relief due to or during loss of consciousness.


    Central depression causing stupor where the animal is unaware of its surroundings but still responsive to painful procedures.

    Surgical Plane of Anesthesia

    The stage of anesthesia in which the animal is at an appropriate anesthetic depth and the surgical procedure can begin.

  • Procedures

    1. Prior to Anesthetic/Analgesic/Sedative Event

    1. Newly arrived animals should have an acclimation period of at least 3 days prior to anesthesia or sedation.
    2. Age and body weight should be considered when designing an A/A plan.
    3. Pre-anesthetic fasting is usually not necessary in rodents. If pre-anesthetic fasting is required:
      1. The fasting period must be limited to 2-3 hours and no longer due to the higher metabolism in mice.
      2. Water should NEVER be restricted.
    4. Apply sterile non-medicated ophthalmic ointment to the eyes to prevent corneal drying during anesthesia or sedation.

    2. Routes of Administration

    1. More detailed information regarding injection techniques and maximum quantities safely administered to mice can be found in Guidelines on Administration of Substances to Laboratory Animals.

    3. Normal Monitoring Parameters

    1. More information on anesthetic/sedation monitoring requirements can found in Anesthesia and Sedation Monitoring Guidelines.
      1. Respiratory rate should be 55 - 100 breaths/min.
        1. A drop in respiratory rate of 50% can be normal during anesthesia.
        2. Respiratory pattern can be used to monitor anesthesia.
          1. Deep and <55 breaths/min: the animal may be too deep.
          2. Shallow and >100 breaths/min: the animal may be too light.
      2. Pulse rate should be 300 - 500 beats/min.
      3. Normal temperature ranges while under anesthesia fall between 36.0°C and 38.0°C (96.8°F – 100.4°F).
      4. Mucus membrane color should be pink. Never pale white or blue.
        1. Normal capillary refill time (CRT) is < 2 seconds.

    4. Recovery

    1. Visibly observe and monitor every 15 minutes during recovery from anesthesia until the animal is fully ambulatory.
      1. Animals that received alpha-2 agonists (dexmedetomidine, xylazine) as part of anesthetic protocol can receive a reversal agent to expedite recovery.
        1. See Table 6 below for recommended doses.
      2. Recover rodents:
        1. Individually until fully ambulatory to avoid cannibalism by cage mates; the recommended method.
        2. In clean cages without bedding to limit the possibility of tracheal foreign body obstruction or aspiration pneumonia.
      3. Monitoring parameters and thermal supplementation should be continued throughout the recovery period.
        1. See Anesthesia and Sedation Monitoring Guidelines and Guidelines on the Performance of Surgery in Rodents for more information on post-operative monitoring and appropriate thermal support devices.
    2. Maintain animals in the surgery area to facilitate appropriate monitoring during the recovery period.
    3. Maintain support following anesthesia as soon as the animal is recovered to facilitate the recuperation process.
      1. Moist chow, regular chow, or diet gel should be provided on the cage floor to encourage eating as soon as possible.

    5. Anesthetics

    1. Detailed information on all approved anesthetics and sedatives can be found in Anesthesia and Analgesia Drug Descriptions.
    2. Table 1: Anticholinergics 
      Drug Dosage and Route 
      Atropine 0.04 - 0.10 mg/kg SC
      Glycopyrrolate 0.01 - 0.02 mg/kg SC
       a Subcutaneous (SC)   
    3. Table 2: Inhalant Anesthetics Used in Mice





         To effect, typically:  
         4 - 5% for induction   
         1 - 2% for maintenance   

         Requires use of a calibrated vaporizer


         300µL on gauze   
         placed in a 500ml   

      • Chamber induction for brief anesthesia for procedures
      • Gauze must be protected so animal cannot come into contact with isoflurane 
      • Use a container that allows animals to be visible during induction
      • Use a container made of a material that can be sanitized 


         To effect, typically:  
         4 - 7% for Induction   
         2 - 4% for maintenance   

         Requires use of a calibrated vaporizer

    1. Table 3: Injectable Anesthetics Used in Mice


         Dosage and Route    

         Duration of   






         + Xylazine   

         80 - 120 mg/kg ket. IP   
         + 5 - 10 mg/kg xyl. IP   

         30 - 45 minutes   

         Re-dose with 1/3 of Ketamine dose   

         + Xylazine   
         + Acepromazine   

         80 - 100 mg/kg ket IP   
         + 5 - 10 mg/kg xyl IP   
         + 1 mg/kg ace IP   

         40 minutes   

         Re-dose with 1/2 of Ketamine dose,   
         or 1/4 of ketamine dose & 1/4 xylazine dose   

         + Dexmedetomidine   

         50 - 75 mg/kg IP   
         + 0.5 - 5 mg/kg IP   

         20 - 30 minutes







         30 - 40 mg/kg IP sedation   
         40 - 60 mg/kg IP anesthesia   

         10 - 300 minutes   

         Respiratory depression /   
         poor analgesia   






         12 - 26 mg/kg IV   

         5 - 7 minutes   

         Titrate as needed   

       a Intraperitoneal (IP), Intravenous (IV)
    1. Table 4: Injectable Sedatives Used in Mice


         Dosage and Route    

         Duration of   


         + Diazepam   

         100 mg/kg ket IP   
         + 5 mg/kg dia. IP   

         20 - 30 minutes   

         Sedation /   

         + Midazolam   

         100 mg/kg ket IP   
         + 5 mg/kg mid. IP   

         20 - 30 minutes   


         + Acepromazine   

         100 mg/kg ket IP   
         + 5 mg/kg ace IP   

         20 - 30 minutes   



         100 - 200 mg/kg IP   


         Poor muscle relaxation   
         / mild analgesia   

       a Intraperitoneal (IP)
    1. Table 5: Injectable Anesthetics Requiring Scientific Justification and IACUC Approval


         Dosage and Route    

         Duration of   



         114 mg/kg IP   


         5% concentration   
         Non-survival procedures   

         Chloral hydrate   

         400 mg/kg IP   


         Non-survival procedures   


         125 - 250 mg/kg IP   

         30 - 45 minutes   

         Avoid re-dosing   

       a Intraperitoneal (IP)
    1. Table 6: Injectable Reversal Agents


         Dosage and Route    

         Reversal Agent For   



         0.5 - 1.0 mg/kg IP, IM or SC   

         or Xylazine   

         Preferred reversal agent for   
         Alpha-2 agonists   


         1.0 - 2.0 mg/kg IP or SC   


         Less effective than Atipamezole   

       a Intraperitoneal (IP), Intramuscular (IM), Subcutaneous (SC)

    6. Analgesia

    1. Unrelieved pain can have profound negative physiologic consequences, which may alter research results.
      1. Mice show a variety of responses to pain, some of which may be fairly subtle and easily missed on casual examination.
      2. Pain evaluation in mice consists of evaluating behavioral and physiologic parameters (see Table 7). 
    2. The IACUC requires preemptive analgesia (analgesics given prior to the first skin incision) unless otherwise justified in the protocol.
      1. Requirements for analgesic coverage differ depending on the classification of surgery as Type I, II, or III.
        1. See the Policy on Analgesia in Animals Undergoing Surgery for updated surgical classifications and analgesia requirements.
      2. The use of buprenorphine as a pre-emptive analgesic may decrease the amount of required anesthetic drugs, due to buprenorphine's sedative and respiratory depressant effects.
    3. See table 8 for mice analgesics.
    4. Table 7: Pain Evaluation Parameters

         Behavioral Signs   

         Physiologic Indicators   

         Reluctance to move   

         Elevated blood pressure   

         Hunched posture   

         Elevated heart rate   

         Social isolation   

         Elevated respiratory rate   

         Decreased appetite   

         Changes in body temperature   

         Decreased grooming   

         Dilated pupils   





         Decreased nest building (see Appendix A)   


         Facial expressions (see Appendix A)   


    1. Table 8: Analgesics Used in Mice


         Dose a   



         0.05 - 0.1 mg/kg SC or IP   

         4 - 8 hours

         Buprenorphine extended-release 
            (Ethiqa XR®)  

         3.25 mg/kg SC   

         48 hours   


         5 mg/kg SC or IP   

         24 hours   


         2.5 mg/kg SC   

         12 - 24 hours   


         1 - 2 mg/kg PO or SC   

         12 - 24 hours   


         2 - 5mg/kg SC   

         12 - 24 hours   

       a Subcutaneous (SC), Intraperitoneal (IP), Oral (PO)

    7. Local Anesthetics

    1. Lidocaine and bupivacaine are the two most commonly used local anesthetics
    2. Table 9: Local Anesthetics Used in Mice


         Dosage and Route a   

         Duration of   



         4 mg/kg SC (0.4 ml/kg of a 1% solution)   

         1.5 - 2 hours   

         Rapid onset (1 - 2 min)   


         1 - 2 mg/kg SC (0.4 - 0.8 ml/kg of a 0.25% solution)   

         4 - 12 hours

         Slower onset (5 - 10 min)   

       a Subcutaneous (SC)
    1. These doses can be diluted in sterile water to provide a larger injection volume. These injectable anesthetics are most routinely administered in subcutaneous tissues near the site of the incision to be made.
      1. Administration can be performed in a "line block," in which the subcutaneous tissue proximal to the incision site is infiltrated with anesthetic in a linear fashion.
      2. Administration can be performed in a "ring block," where subcutaneous tissue around the incision site is infiltrated circumferentially.
    2. High plasma concentrations of lidocaine or bupivacaine can cause cardiovascular effects (e.g., hypotension, dysrhythmias) and central nervous system depression followed by seizures. To avoid these adverse consequences:
      1. Weigh each animal individually and only give the maximum safe dose calculated for that individual.
    3. Aspirate the syringe prior to injection to ensure that IV injection is avoided.
    4. Local anesthetics are available in a variety of concentrations with or without epinephrine. Epinephrine causes vasoconstriction and prolongs the action of the local anesthetic. Epinephrine should not be used in animals are suspected to have compromised cardiac function, or in locations that have poor collateral blood flow (distal tail, paw, etc.).

    8. Neonatal Rodent Anesthesia

    1. A mouse neonate is defined as < 10 days of age. There are several anesthetic methods currently presented in the literature for use in neonatal rodents and include:
      1. Injectable
      2. Inhalant
      3. Physical methods
    2. Injectable anesthetics have been associated with a high mortality in neonatal rodents. Do not use injectable anesthetics in neonatal mice <7 days old.
    3. Inhalant anesthetics in neonatal rodents have been associated with longer induction and recovery times than adult rodents with inhalant anesthetics.
    4. Hypothermia is the primary physical method utilized in neonatal rodent anesthesia.
      1. It is believed to provide anesthesia/analgesia by decreasing neural conduction and synaptic transmission.
      2. Hypothermia can only be performed in neonatal rodents <6 days old and should not be used for procedures lasting longer than 30 min.
      3. Ensure a barrier is between the neonate and the cooling agent (e.g. bed of crushed ice or chilled cold pack) at all times to prevent direct damage to the tissues; examples include:
        1. Placing a latex covering over the cooling agent.
        2. Placing the neonate in a cut off finger of a latex glove.
        3. Placing the neonate in a paper-lined test tube.
      4. Check the neonate for pedal reflex indicating proper plane of anesthesia.
      5. Maintain the neonate on a cooling agent for the procedure.
      6. Use fiber optic lighting for the surgical field as incandescent bulbs may warm the neonate.
      7. Re-warm the neonate slowly following hypothermia anesthesia.
        1. Rapid warming can cause tissue damage.
        2. Use of a circulating water heating pad set at 40 ºC (104ºF) or in an incubator set at 33 ºC (91.4ºF) is recommended.
      8. Return neonate to dam once are able to crawl.
    5. Parental cannibalism can occur with neonates after anesthesia. Follow the below steps to reduce the occurrence of cannibalism in anesthetized neonates, if possible:
      1. Ensure the neonate is fully recovered before returning to the dam.
        1. Smear the neonate with soiled bedding from the mother's cage.
    6. Place the neonate back in the middle of the litter
    7. Table 10: Inhalant Anesthetics in Neonatal Mice

         Stage of   



         Isoflurane (%)   


         Mask or Chamber   

         0.5 - 1   

         4 - 5   



         0.5 - 1   

         1 - 2   

       Note: Neonates typically require a higher inhalant anesthetic dose than that observed in adults.
    1. Table 11: Injectable Anesthetics in Neonatal Mice


         Dosage and Route   

         Duration of Action   


         + Xylazine   

         50 - 150 mg/kg (K)   
         + 5 - 10 mg/kg (X)   
         IP a or SC b   

         20 - 40 minutes   

         Only to be used in mice   
         greater than 7 days old   

       a Intraperitoneal (IP): 27g needle, 1 ml syringe;, maximum volume 0.5ml
       b Subcutaneous (SC): 27g needle, 1 ml syringe, maximum volume 1 ml

    9. Neonatal Rodent Analgesia

    1. There is little information regarding the efficacy and dose ranges for the use of analgesics in neonates.
    2. Some data suggest that unalleviated pain in neonates can alter responses to pain and stress later in life.
    3. Investigate the literature on neonatal analgesia when a project involving neonatal surgeries and procedures is started, if possible. (LaPrairie and Murphy, 2010; Sternberg et al., 2005; Victoria et al., 2013a,b, 2014; LaPrairie et al., 2008; Walker et al., 2009)

    10. Emergency Resuscitation

    1. Respiratory depression can be treated by the administration of doxapram 5-10mg/kg IV or IP.
      1. If respiratory depression reoccurs, the doxapram should be administered repeatedly at approximately 10-15 minute intervals.
    2. Supportive care for animals which reach too deep a level of anesthesia includes:
      1. Decreasing or discontinuing inhalant anesthetic.
      2. Stimulating the animal by gentle manipulation of the body.
      3. Raising the body temperature to normal.
      4. Providing supplemental oxygen through a facemask or nose-cone.
    3. Administering reversal agents of anesthetic drugs, if applicable. See Table 6 above.
  • Appendix A: ULAM Assessment of Pain in Mice Handout

  • References

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    42. Websites regarding neonatal anesthesia:
Species: Mice

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