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Guidelines
Suggested parameters and sets of instructions outlining best practices and standards for accomplishing specific animal care and use research duties.

Guidelines on Anesthesia and Analgesia in Mice

Unit for Laboratory Animal Medicine

| Approval Date:

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.

Anesthesia

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.
Related Terms: Analgesia, A/A, Local Anesthetic, Surgical/Anesthetic/Sedation Record

Analgesia

Provides pain relief without loss of consciousness.

Related Terms: A/A, Multimodal Analgesia, Pre-emptive Analgesia, Systemic Analgesic, Anesthesia

A/A

Anesthesia and analgesia.

Related Terms: Anesthesia, Analgesia

Sedation

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.

Related Terms: Anesthesia, Local Anesthetic

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 the 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 Routea
    Atropine 0.04 – 0.10 mg/kg SC
    Glycopyrrolate 0.01 – 0.02 mg/kg SC

    aSubcutaneous (SC)

  3. Table 2: Inhalant Anesthetics Used in Mice

    Drug Dosage Comments
    Isoflurane
    Recommended    		 To effect, typically:
    4 – 5% for induction
    1 – 2% for maintenance    		 Requires use of a calibrated vaporizer
    Isoflurane 300µL on gauze placed in a 500ml container
    • 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
    Sevoflurane 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

    Drug Dosage and Routea Duration of Anesthesia Comments
    Dissociatives
    Ketamine
    + Xylazine
    Recommended    		 80 – 120 mg/kg ket. IP
    + 5 – 10 mg/kg xyl. IP    		 30 – 45 minutes Re-dose with 1/3 of Ketamine dose
    Ketamine
    + 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
    Ketamine + Dexmedetomidine 50 – 75 mg/kg IP
    + 0.5 – 5 mg/kg IP    		 20 – 30 minutes
    Barbituates
    Pentobarbital 30 – 40 mg/kg IP sedation
    40 – 60 mg/kg IP anesthesia    		 10 – 300 minutes Respiratory depression / poor analgesia
    Other
    Propofol 12 – 26 mg/kg IV 5 – 7 minutes Titrate as needed

    aIntraperitoneal (IP), Intravenous (IV)

  1. Table 4: Injectable Sedatives Used in Mice

    Drug Dosage and Routea Duration of Anesthesia Comments
    Ketamine
    + Diazepam    		 100 mg/kg ket IP
    + 5 mg/kg dia. IP    		 20 – 30 minutes Sedation / immobilization
    Ketamine
    + Midazolam    		 100 mg/kg ket IP
    + 5 mg/kg mid. IP    		 20 – 30 minutes Immobilization
    Ketamine
    + Acepromazine    		 100 mg/kg ket IP
    + 5 mg/kg ace IP    		 20 – 30 minutes
    Ketamine 100 – 200 mg/kg IP Unproven Poor muscle relaxation / mild analgesia

    aIntraperitoneal (IP)

  1. Table 5: Injectable Anesthetics Requiring Scientific Justification and IACUC Approval

    Drug Dosage and Routea Duration of Anesthesia Comments
    Alpha-chloralose 114 mg/kg IP Unproven 5% concentration
    Non-survival procedures
    Chloral hydrate 400 mg/kg IP Unproven Non-survival procedures
    Tribromoethanol 125 – 250 mg/kg IP 30 – 45 minutes Avoid re-dosing

    aIntraperitoneal (IP)

  1. Table 6: Injectable Reversal Agents

    Drug Dosage and Routea Reversal Agent For Comments
    Atipamezole 0.5 – 1.0 mg/kg IP, IM or SC Dexmedetomidine or Xylazine Preferred reversal agent for Alpha-2 agonists
    Yohimbine 1.0 – 2.0 mg/kg IP or SC Xylazine Less effective than Atipamezole

    aIntraperitoneal (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 pre-emptive 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
    Aggression
    Self-mutilation
    Decreased nest building (see Appendix A)
    Facial expressions (see Appendix A)
  1. Table 8: Analgesics Used in Mice

    Drug Dosea Duration
    Buprenorphine 0.05 – 0.1 mg/kg SC or IP 4 – 8 hours
    Buprenorphine extended-release (Ethiqa XR®) 3.25 mg/kg SC 48 hours
    Carprofen 5 mg/kg SC or IP 24 hours
    Flunixin 2.5 mg/kg SC 12 – 24 hours
    Meloxicam 1 – 2 mg/kg PO or SC 12 – 24 hours
    Ketoprofen 2 – 5mg/kg SC 12 – 24 hours

    aSubcutaneous (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

    Drug Dosage and Routea Duration of Anesthesia Comments
    Lidocaine 4 mg/kg SC (0.4 ml/kg of a 1% solution) 1.5 – 2 hours Rapid onset (1 – 2 min)
    Bupivacaine 1 – 2 mg/kg SC (0.4 – 0.8 ml/kg of a 0.25% solution) 4 – 12 hours Slower onset (5 – 10 min)

    aSubcutaneous (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 Anesthesia Route Oxygen (L/min) Isoflurane (%)
    Induction Mask or Chamber 0.5 – 1 4 – 5
    Maintenance Mask 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

    Drug Dosage and Route Duration of Action Comments
    Ketamine
    + Xylazine    		 50 – 150 mg/kg (K)
    + 5 – 10 mg/kg (X) IPa or SCb    		 20 – 40 minutes Only to be used in mice greater than 7 days old

    aIntraperitoneal (IP): 27g needle, 1 ml syringe;, maximum volume 0.5ml
    bSubcutaneous (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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SPECIES: Mice

TOPICS: Anesthesia and Analgesia

Questions?

If you have questions or comments about this document, contact ULAM Veterinary Staff ([email protected]).

The ULAM Training Core ([email protected] or 734-763-8039) can be contacted to provide training in techniques at no charge.

For any concerns regarding animal health after work hours or on holidays/weekends, contact DPSS (3-1131) who will contact the on-call veterinarian.