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Anaesthesia of laboratory animals Timo Nevalainen University of Eastern Finland Terminology Anaesthesia = without sensation an = without, aestos = sensation Analgesia = without pain an = without, algios = pain Euthanasia = good death

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Anaesthesia of laboratory animals

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anaesthesia of laboratory animals

Anaesthesia of laboratory animals

Timo Nevalainen

University of Eastern Finland

  • Anaesthesia = without sensation
    • an = without, aestos = sensation
  • Analgesia = without pain
    • an = without, algios = pain
  • Euthanasia = good death
    • eu = good, thanatos = death
how anaesthesia is chosen
How anaesthesia is chosen ?
  • Tradition
    • look articles of your discipline
  • Ask colleagues
    • end up to tradition
  • Rational way ?
anaesthesia how
Anaesthesia, how ?
  • Choice of anaesthetic
    • minimal interference of study
    • immobilisation and no/less pain
    • nature of the procedure
    • duration of the procedure
  • Humane handling of animals and safety of personnel are important
problems with laboratory animals
Problems with laboratory animals
  • Group anaesthesia
  • Large species, strain etc. differences
  • Follow-up difficulties
  • Anaesthesia poorly known
  • Clinical and research anaesthesia are not used properly
  • Postoperative care does not work
pros and cons of anaesthesia

no pain

no muscle reflexes

no muscle tension

Clinical anaesthesia


changes in the body

physiological status different

responses may be different

Research anaesthesia

Pros and cons of anaesthesia
anaesthesia vs cns mediated reflexes
Depresses least

depresses most


thiobarbiturate + N2O






Anaesthesia vs. CNS-mediated reflexes
inspection before anaesthesia
Inspection before anaesthesia
  • Check animals before starting
  • Do not anaesthetise sick animals, they are unsuitable for experiments anyway
  • Pay attention to symptoms of infections
bedding volatile compounds with highest sums
Bedding Volatile Compounds / With Highest Sums

concentration microg/g

Vesel et al. 1966: Bedding volatile compounds induce liver microsomal enzymes

how to fast before anaesthesia
How to fast before anaesthesia ?
  • Take away food - not water
  • the smaller species, the faster metabolism, the shorter fasting
  • no fasting for mouse
  • abdominal procedures in rat, fast for 6 h
  • guinea-pig 6 h
  • rabbit 6 h
dangers of vomiting
Dangers of vomiting
  • Horse vomits = rupture of stomach
  • ruminants can drop ruminating balls
  • carnivores vomit easily
  • among rodents guinea-pig vomits easily
  • can drain into trachea and cause aspiration pneumonia
handling vomiting dangers
Handling vomiting dangers
  • Fasting decreases amount vomited, but reflex stays
  • if vomits, place head over table edge, drains away from mouth
  • once anaesthetised, intubate, closes passage to respiratory system
pre anaesthetic hydration
Pre-anaesthetic hydration
  • Give animal balanced electrolyte fluid to drink couple of days before
  • yields better water balance
  • continue a few days after anaesthesia (and procedure)
pre medication atropine
Pre-medication atropine
  • No saliva, no vagal reflexes, less gut motility
  • Dose: rodents 0.05 mg/kg, rabbit 1-3 mg/kg about 30 min before
  • if autonomic nervous system is involved, atropine may be contraindicated
sedative pre medication
Sedative pre-medication
  • Decrease dose of anaesthetic by 20-50 %
  • better handling may be needed eg for iv injection
  • if procedure is not painful, but immobilisation is needed
  • some compounds dilate vessels - easier to see them
ensuring oxygenation
Ensuring oxygenation
  • Respiratory passages open
    • posture, atropine
  • additional oxygen
    • tubing directly into mouth
  • less dead space
    • intubation
control of anaesthesia
Control of anaesthesia
  • Inhalation is well controlled
  • iv-bolus anaesthesia - you give half of calculated dose, the rest to effect
  • infusion anaesthesia also well controlled
  • im, ip ja sc administration: you give calculated bolus - response may be variable
  • Mouse - tracheostomy
  • rat - tube outer diameter = 1-1.5 mm, length 2 cm attached snugly inside wider tube
  • rat placed on back, fixed by maxilla incisors, tongue pulled out
  • larynx visible, becomes easier with high intensity light directed to neck
  • Guinea pig intubation - easier version of rat, tube outer diameter =2 - 2.5 mm
  • Rabbit intubation difficult
    • laryngospasm unless not deep enough
    • small place, otoscope / pediatric laryngoscope
    • tube outer diameter = 3 - 3.5 mm, no cuff
  • Rabbit: guided or blind intubation
assessment of anaesthetic depth video
Assessment of anaesthetic depth (video)
  • Mouse
    • respiratory rate, cornea
    • tail pinch and pedal reflex
    • pedal best
  • Rat
    • respiratory rate, tail pinch
    • pedal reflex and ear pinch
    • ear pinch best
anaesthetic depth
Anaesthetic depth
  • Guinea-pig
    • palpebral reflex and ear pinch
    • ear pinch best
    • may move 1-2 times, is not lightening of anaesthesia
  • Rabbit
    • light surgical - pedal reflex
    • medium depth - palpebral reflex & ear pinch
    • corneal reflex - dangerously deep
dose memos
Dose memos
  • Rabbit dose calculator
hypnorm midazolam
Hypnorm & midazolam
  • clinical anaesthesia, reasonable safety margin
  • NOT to be given ip, liver metabolism weakens effect
  • contains fentanyl = controlled substance
  • recovery is speeded by nalorphin
  • Rat: Combination 0.15 - 0.2 ml / 100 g sc
  • Mouse: Combination 0.10 - 0.15 ml / 20 g sc
hypnorm midazolam38
Hypnorm – Midazolam
  • Combination
    • One part HypnormR (fentanyl-fluanisone) + one part midazolam (DormicumR, 5 mg/ml) + two parts sterile water
    • Mix both drugs first with water and then combine. Do not keep in refrigerator.
    • Duration of anesthesia: Mouse 30-60 min, rat 20-90 min
    • Reversal: Nalorphine 1 mg / kg iv, im, ip
medetomidine fentanyl
Medetomidine & fentanyl
  • Works reasonably well in rats
  • animals pale and urine drips
  • major advantage: antagonist wakes them real fast
  • Common in neuropharmacology
  • if too concentrated, fatal paralytic ileus, abdomen dilated, do badly and die
  • correct concentration is 36 mg/ml or less
infusion anaesthesia
Infusion anaesthesia
  • Typical compound propofol
  • used in rats, single bolus 10 mg/kg produces anaesthesia of about 5 min
  • infusion anaesthesia can provide stable anaesthetic level without a vaporizer
barbiturate anaesthesia
Barbiturate anaesthesia
  • Safety margin in rabbits narrow
    • may lead to 20-40 % mortality
    • if used - only for terminal procedures
  • Mouse - the same situation
    • can combine with e.g. ethanol
    • there are better combinations
the univentor 400 anaesthesia unit

designed to control the mixture of anesthetic and air with the precision required to successfully operate on animals weighing from 20-500 grams

  • halothane
    • common inhalation compound
    • does not evaporate concentrations with mortality at room temperature
    • liver necrosis
    • cheapest of proper inhalation compounds
    • good clinical anaesthesia with guidance
  • Combines reasonable research anaesthesia and good guidance
  • more expensive than halothane
  • requires own vaporizer
  • no mortal concentrations at room temperature
no ether neither chloroform
No ether, neither chloroform
  • Evaporate to mortal concentrations at room temperature
  • ether explodes, and carcasses in cooler of freezer smell a long time
  • chloroform is liver toxic and suspected carcinogen
  • Useful for 1-2 min procedures, no longer
  • for rats and mice
  • incubation box with animals, tube CO2 in at a rate 0.6 x box volume l/min
  • righting reflex disappears, move to mouth cone with 50 % CO2 and 50 % O2
postoperative care
Postoperative care
  • temperature
    • infrared light bulb
    • insulation
  • fluid
    • ip or sc as boluses
    • air humidification
  • additional oxygen
    • carbogen flow to chamber
strategy for research anaesthesia
Strategy for research anaesthesia
  • Simple is best - avoid polypharmacy
  • Effect on your study
  • Prefer inhalation
    • stable anaesthesia
    • can measure inhalant concentration from expired air = best description of anaesthesia
  • Unpleasant sensory or emotional feeling, may / may not be associated with real or potential damage
  • It is reasonable to assume that animals feel pain
  • The mechanisms behind are similar
animal pain
Animal pain
  • may not be identical to humans
  • it is assumed that intensity and duration of pain is variable in specific tissue damage between species
  • fast recovery leads to underestimation of pain and failure to give analgesic
why pain
Why pain?
  • It is a warning signal
    • specific part of body will not be used
  • Pain can also be harmful
    • lack of use & spasms -> weakness, loss of muscle and permanent change
  • Pain can result in lack of appetite and drinking
pain assessment
Pain assessment
  • difficult in humans even though we understand what they say
  • animal approaches
    • anthropomorphic way
    • based on clinical findings
      • activity, grooming, immobility, vocalization, posture, aggressiveness
pain assessment61
Pain assessment
  • rodents are nocturnal
  • animals should not notice
    • infrared lamp during dark
  • handling responses
signs of pain
Signs of pain
  • Species and procedure specific
  • Scoring system is a necessity
    • analgesic treatment
    • verification of analgesia efficacy
  • postoperative analgesia the most common form
  • humans with neoplasia and infections receive analgesia
    • fear for interference in animal studies
difficulty of assessment
Difficulty of assessment
  • Animal shows no sign of pain
    • pain will not be treated
  • Comparison to human situation
    • better to give a single dose
    • repeated dose may be problematic
      • e.g. appetite may be lost -> recovery delayed
pain scoring requires
Pain scoring requires
  • knowledge of
    • species specific behavior
    • behavior before the procedure
  • palpation of the tissue & response
  • evaluation of sore area (e.g. leg)
  • knowledge on efficient analgesics doses, and possible behavioral consequences
ideal analgesic
Ideal analgesic
  • Does not interfere the study
  • No sedation
  • Long duration
  • Efficient analgesia
analgesia contraindications
Analgesia; contraindications
  • Analgesia does NOT replace
    • good surgical technique
    • good peri- and postoperative care
  • Analgesia should not interfere with the study or interpretation of the results
  • systemic analgesia
    • duration?
  • local analgesia
    • applied on the incision area
  • Rat (R) and Mouse (M)
    • Opioids - look for duration
  • Buprenorphine
    • 0.05(R), 0.1(M) mg/kg sc / 6-12 h
  • Butorphanol 2.0(R) mg /kg sc / 1-2 h
  • Petidine 10-20(R&M) mg/kg sc or im / 3 h
  • Morphine 2-5(R&M) mg/kg sc /4 h
  • Rat (R) and Mouse (M) doses
    • many given per os / dissolve in water
  • Carpofen 5(R&M) mg/kg sc or per os
    • R 12-24h
  • Fluniksine 2.0(R) mg/kg sc /1-2h
  • Ketoprofeine (R) 5 mg/kg im/12-24h
analgesia advantages
Analgesia - advantages
  • Faster recovery
  • Faster return of appetite
  • No weight loss
  • Which is better:
    • effect of pain and procedure or only effect of procedure
  • Ethically right
how to use
How to use ?
  • Small surgical procedures -> single injection e.g. buprenorphine
  • in major interventions continue 2-3 days
  • Additionally place local analgesia into the incision
  • Follow animals and verify efficacy
pain assessment after laparatomy in rats
Pain assessment after laparatomy in rats
  • Stage 1: Visual Analogue Score (VAS)
    • assess pain experienced by the rat with a mark on a 10 cm line
    • there are 4 clips to assess
  • Stage 2: Instructions
  • become familiar with behaviors typical to pain
    • do not count at this point
  • Only three behaviors needs to be recognized
    • order haphazard like in real life
pain behaviors 1
Pain behaviors 1
  • Twitches
    • usually when the rat rests
    • most common on back, fur coat movement
    • also on the head
    • most common behavior
  • Back arching
    • feet extended, belly goes up, arching back
    • often just prior to walking
pain behaviors
Pain behaviors..
  • Falling
    • temporary loss of balance
    • falls to side or backwards
  • Each of all three are counted as one
  • P.Flecknell. Laboratory Animal Anaesthesia. Academic Press, 1996
  • H.B. Waynforth & P.A. Flecknell: Experimental and Surgical Technique in the Rat. Academic Press 1992
  • D.H. Kohn, S.K. Wixson, W.J. White, G.J. Benson. Anesthesia and Analgesia in Laboratory Animals. Academic Press 1997.