Anesthesia of Neonatal Mice and Rats

Introduction

Neonates are defined as mouse or rat pups up to 10 days of age.

Gas anesthesia (isoflurane) or hypothermia may be used to anesthetize neonatal rats and mice. Parenteral agents are not advisable in very young animals. Hypothermia allows less control of anesthetic depth compared to inhalant anesthesia. Hypothermia is appropriate for short, minor surgical procedures (5–15 minutes) in neonatal rats or mice up to 5 days of age. Gas anesthesia is recommended for longer, more invasive procedures and in neonates older than 5 days. Very young hairless, altricial rodents function as poikilotherms and are resistant to brain damage associated with cephalic circulatory arrest. Surface cooling is effective due to their small surface area and body size. Hypothermia provides immobilization and mild analgesia (similar to local anesthesia resulting in nerve transmission blockage). For more information, contact veterinary staff for assistance.

Policies

A. Neonates must be anesthetized for painful procedures and surgery.

B. Neonates must receive analgesia if the procedure can reasonably be expected to result in enough pain to interfere with the neonatal function of sleeping and suckling. Exceptions may be considered, if the disturbance of neonate and dam in order to administer the medication interferes more than warranted with these functions.

C. Documentation is required for neonatal anesthesia, surgery, and postoperative care. Please use the IACUC Anesthesia & Surgical Monitoring Form for Rodents or a substantially similar form.

Procedures

A. Recommended Preparation of Dam Prior to Recovery Anesthesia and Surgery on Neonates1. These procedures may help prevent the dam from cannibalizing or neglecting pups.

1. Handling and olfactory conditioning of the dam 7–10 days prior to birthing.

a. Gentle handling for five-minute intervals a few times a day.
b. Cotton balls scented with the chemicals (Betadine, isopropyl alcohol, Chlorhexidine, etc.) or gas anesthetic (isoflurane) to be used placed in cage for 1–2 minutes daily.
c. Never leave animal unattended during this time3.

B. Hypothermia1
1. Protect pup with glove or by placing in a paper-lined test tube to avoid freeze damage to the skin. A thin material, such as a latex glove, works better than a thicker material (e.g., a neoprene glove).
2. Immerse container holding pup in ice water or place on a barrier such as a glove or other waterproof material over crushed ice. If crushed ice is used, it must be carefully packed to provide close contact between ice and pup. The ice can be packed flat or a frozen block of ice covered with a thin lining can be used.
3. Induction time: 2–4 minutes. Observe the pup closely.
4. Anesthesia time: 5–15 minutes.
5. Recovery time: Variable (up to one hour).
6. The pup can be kept on a cooling platform (i.e., waterproof material-covered ice cube or cold glass plate or a frozen ice block) to provide more constant level of hypothermia.
7. Use of a fiber-optic surgical lamp rather than an incandescent one minimizes surface warming and helps in maintaining hypothermia.

C. Recovery of Neonate from Hypothermia
Recover the neonate by holding it in your gloved hand or placing it in a petri dish on a warm water-circulating heating pad or under a heat lamp for less than five minutes (not too close to the lamp), watching carefully to avoid overheating.

D. Preparing the Neonate for Return to the Dam
1. Recover neonate on home cage/dam bedding so as to make it smell similar to what it did before removal from cage.
2. Make sure neonate is warm and pink and breathing and capable of spontaneous movement before returning it to the dam.
3. Returning neonates as a group (the entire litter) is best to minimize disturbing the dam.
4. If isoflurane anesthesia has been used, it may help to accustom the dam to the smell for 1–2 days before surgery on the neonate (see section A above).3
5. It may help the dam accept pups back after a procedure if all vestiges of blood and disinfectant are removed from the pups; gently wipe them with a wet gauze pad before returning them to the dam.4

E. INHALANT ANESTHESIA

Isoflurane—Method
Inhalant anesthesia may be safe both for ultra-short (<5 minutes) and prolonged (>6 hours) procedures. For longer periods of anesthesia, it may be necessary to ventilate the animal.2,6,7
1. Induce anesthesia in an induction box.
2. Use an adult mouse face mask adjusted for neonate, or use the EZ-103A Microflex Breather and shave off a small amount to shorten the mask to fit the neonate.5
3. Administer isoflurane at 1.5–2.5% in 100% oxygen.
4. Place neonate on a warm pad or circulating warm-water pad.
5. During the recovery phase, turn off inhalant gas before oxygen.

INHALANT ANESTHESIA (Isoflurane) FOR NEONATAL RATS AND MICE

Phase of Anesthesia Oxygen L/min Isoflurane %
Induction 0.5–1.0 4–5
Maintenance 0.5–1.0 1–2
Recovery 0.5 then to 0* 0

*During the recovery phase, turn off inhalant gas before oxygen.

F. Parenteral (Injectable) Anesthetics
A ketamine/xylazine cocktail may be considered for use in neonates but may not be as safe as hypothermia or isoflurane. It is extremely important to accurately weigh the animal and calculate the correct dose. Starting with the lower dose range is recommended. Only the IP route of administration is allowed. Recovery period may be prolonged. Repeat administration is not advisable. Hypothermia must be avoided by keeping the neonate warmed as described above in section C.

Injectable Anesthetic Agents for Neonatal Rats and Mice
Do not use in pups <7 days old.

Drug Species Dose mg/kg Route and needle size (gauge) Max. volume
ml
Duration
minutes
Ketamine/Xylazine MOUSE 50–150/5–10 IP <27g <0.5 20–40
Ketamine/Xylazine RAT 40–90/5–10 IP <25g <1.0 20–30

G. PREEMPTIVE AND/OR POSTOPERATIVE ANALGESIA

ANALGESIC AGENTS FOR NEONATAL RATS AND MICE

Begin by administering a lower dose than would be administered to adult animals. These opioids have been shown to be safe and effective analgesics in neonatal rodents3.

*Handling of neonates should be minimized. See I.B.

Drug Species Dose mg/kg Route and needle size (gauge) Max. volume
ml
Frequency*
Buprenorphine MOUSE 0.05–0.5 IP or SC <27g <0.2 Every 6–8 hours
Buprenorphine RAT 0.01–0.05 IP or SC <25g <0.2 Every 6–8 hours
Morphine MOUSE 5–10 Every 4 hours
Morphine RAT 5–10 Every 4 hours

Acetaminophen (0.1ml PO of cherry-flavored Children’s Tylenol) at 80mg/2.5ml once or twice a day may be acceptable and can be administered using a dropper or TB syringe1.

References

1. McGill University. University Animal Care Committee (UACC). Neonatal Rodent Survival Surgery.
2. Loepke, AW et al. The physiologic effects of isoflurane anesthesia in neonatal mice. Anesth.Analg. 2006, Jan; 102(1):75–80.
3. Gaertner et. al. Anesthesia and Analgesia in Neonatal Mice and Rats. In Anesthesia and Analgesia 2nd Ed. PP.277–278.
4. Biomethodology and Surgical Techniques in The Mouse in Biomedical Research. 2007. Fox, James G. et.al. Eds.
5. E-Z Anesthesia http://www.ezanesthesia.com/ez_anesthesia/index.html. Go to “Breathing Devices” EZ-103A Microflex Breather.
6. Loepke, AW et.al. The effects of neonatal isoflurane exposure in mice on brain cell viability, adult behavior, and memory. Anesth.Analg. 2009. Jan; 108(1):90–104.
7. Istaphanous GK, Loepke, AW: General anesthetics and the developing brain. Curr Opin Anesthesiolol. 2009 Jun; 22(3):368–73.

Recommended Literature

1. Sheldon, R. Ann et.al. Feb. 2008. Genetic and pharmacologic manipulation of oxidative stress after neonatal hypoxia-ischemia. Int. J. Dev. Neurosci. 26(1): 87–92.
2. Wiesmann, Frank et.al. February 2000. Developmental changes of cardiac function and mass assessed with MRI in neonatal, juvenile, and adult mice. Am J Physiol Heart Circ Physiol 278: H652–H657.

BU IACUC Approved January 2011; Revised January 2014