IACUC Policy and Guidelines on Use of Cryoanesthesia in Neonatal Mice

Overview

Cryoanesthesia is an anesthetic technique in which hypothermia is used to induce anesthesia in neonatal mice. This policy establishes acceptable practices for the use of cryoanesthesia  in neonatal mice undergoing brief surgical procedures.  This policy and guidelines are intended to ensure humane animal care and use consistent with the Guide for the Care and Use of Laboratory Animals1 and current laboratory animal veterinary practice.

Policy

Cryoanesthesia may be used only in neonatal mice through postnatal day 7 (P7; where the day of birth is designated P0) for brief surgical procedures approved by the IACUC.

Mice older than P7 must be anesthetized using an IACUC-approved inhalational or injectable anesthetic regimen appropriate for the procedure.

Scientific Rationale

During the early postnatal period, altricial rodents such as mice have limited thermoregulatory capacity and are more tolerant of controlled cooling than older animals2. Progressive cooling decreases central nervous system activity, producing reversible loss of consciousness and immobilization sufficient for selected brief surgical procedures3.

Hypothermia has been reported as an effective anesthetic method in altricial rodents during the early postnatal period4. Current laboratory animal medicine guidance recommends limiting the use of hypothermia anesthesia to very young rodents (approximately the first postnatal week). Accordingly, the UCI IACUC limits the use of cryoanesthesia to mice through postnatal day 7 (P7). Mice older than P7 must be anesthetized using an IACUC-approved inhalational or injectable anesthetic regimen appropriate for the procedure2.

Appropriate Uses

Cryoanesthesia is appropriate for brief surgical procedures in neonatal mice, including but not limited to:

  • Intracerebral or stereotaxic injections
  • Viral vector administration
  • Tissue transplantation
  • Other brief surgical procedures specifically approved by the IACUC

Procedures anticipated to require prolonged surgical anesthesia should be performed using inhalational or injectable anesthetics.

Procedure

Preparation

An ice-water slurry should be used for cooling.  Crushed ice alone should not be used because it does not provide the same uniform cooling characteristics as ice-water slurry.

Each pup should be placed head-up within a thin protective barrier, such as a finger cut from a non-powdered latex or nitrile glove. This allows for circumferential contact with the cooling medium while preventing direct contact between the skin and ice.

Induction and Maintenance of Anesthesia

Animals should be cooled until loss of spontaneous movement and righting reflex are observed. The time required to achieve this anesthetic state varies depending on the age and size of the pup and the cooling method used, but generally at least 3 minutes4 .

Once an adequate anesthetic state has been achieved, the pup may be transferred to a chilled surgical surface protected by a thin barrier to prevent direct contact with frozen materials. Spontaneous movement and righting reflex should remain absent before beginning the surgical procedure.

If movement is observed in response to initial surgical stimulation, the pup should be returned to the cooling medium until an adequate anesthetic state is re-established.

During the procedure, investigators should provide cooling sufficient to maintain anesthesia while avoiding excessive cooling and unnecessarily prolonged exposure to hypothermia.

Surgical illumination should be selected and positioned to minimize warming of the animal and surgical field. Cool-light sources (e.g., LED or fiber-optic systems) are recommended.

Duration

Cryoanesthesia is appropriate only for short procedures.

Investigators should minimize time under hypothermia consistent with completion of the approved procedure. Procedures expected to exceed approximately 15–20 minutes should be discussed with a veterinarian.

Recovery

Following completion of the procedure, pups should be placed in a warm recovery environment until normal skin color, spontaneous movement, and righting reflex return.

Active warming (e.g., incubator set at next temperature 33oF, a circulating warm-water blanket set to provide 33oF within the cage or other appropriate warming device) may be used.  Rapid warming should be avoided4.

Pups should be returned to the dam only after they are warm, active, and capable of normal movement and nursing.

Animals should be observed after return to the litter for evidence of maternal acceptance and nursing.

Limitations of Cryoanesthesia

Cryoanesthesia shall not be used:

  • in mice older than postnatal day 7 (P7);
  • for procedures anticipated to require prolonged anesthesia;
  • for procedures expected to cause moderate or severe postoperative pain unless appropriate analgesia is provided;
  • when the procedure requires a degree of anesthetic depth control or physiological support that cannot reliably be achieved with cryoanesthesia.

Potential Complications

Potential complications associated with cryoanesthesia include:

  • inadequate anesthetic depth
  • excessive cooling or prolonged recovery
  • delayed return to normal activity
  • localized cold injury from inappropriate contact with ice or frozen surfaces
  • reduced ability to compete for nursing following recovery

Improper recovery may increase the risk of maternal rejection or failure to nurse.

References

  1. Guide for the Care and Use of Laboratory Animals, 8th National Research Council, National Academies Press, 2011.
  2. Blumberg, M.S. and Sokoloff, G. Thermoregulatory Competence and Behavioral Expression in the Young of Altricial Species – Revisited. Developmental Psychobiology. 33(2):107–123, 1998
  3. Flecknell, P. Laboratory Animal Anaesthesia, 4th Edition. Academic Press. P. 137, 2016.
  4. Phifer, C.B., Terry L.M. Use of Hypothermia for General Anesthesia in Preweanling Rodents. Physiology & Behavior 38: 887-890, 1986.