Anaesthesia of laboratory animals

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Anaesthesia of laboratory animals

• Timo Nevalainen
• Universities of Kuopio
• Finland

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A more pharmacologic presentation www.uku.fi/tne
valai/Anesthesia.ppt
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Terminology

• Anaesthesia without sensation
• an without, aestos sensation
• Analgesia without pain
• an without, algios pain
• Euthanasia good death
• eu good, thanatos death

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How anaesthesia is chosen ?

• Tradition
• look articles of your discipline
• Ask colleagues
• end up to tradition
• Rational way ?

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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

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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

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Pros and cons of anaesthesia

• Pros
• no pain
• no muscle reflexes
• no muscle tension
• Clinical anaesthesia

• Cons
• changes in the body
• physiological status different
• responses may be different
• Research anaesthesia

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Anaesthesia vs. CNS-mediated reflexes

• alphachloralose
• thiobarbiturate N2O
• cyclopropane
• barbiturates
• ether
• halothane
• chloroform

• Depresses least
• depresses most

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Inspection before anaesthesia

• Check animals before starting
• Do not anaesthetise sick animals, they are
  unsuitable for experiments anyway
• Pay attention to symptoms of infections

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Bedding Volatile Compounds / With Highest Sums
concentration microg/g
Vesel et al. 1966 Bedding volatile compounds
induce liver microsomal enzymes
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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

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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

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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

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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)

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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

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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

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Ensuring oxygenation

• Respiratory passages open
• posture, atropine
• additional oxygen
• tubing directly into mouth
• less dead space
• intubation

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Pulse oxymeter
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Sensor around leg
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Maintaining normal temp
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ECG-recording
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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

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Inhalation - open mask
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Intubation

• 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

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Intubation

• 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

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Mouse intubation video
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Using otoscope
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Air movement ?
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Rodent respirator
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Wet - loosing heat
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Skin disinfection
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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

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Tail pinch
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Pedal reflex
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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

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Ear pinch
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Dose memos

• www.uku.fi/tnevalai/ratmouseanes.html
• Rabbit dose calculator
• www.morfz.com/rx/drugcalc.html

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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

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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

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Medetomidine fentanyl

• Works reasonably well in rats
• animals pale and urine drips
• major advantage antagonist wakes them real
  fast

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Chloralhydrate

• Common in neuropharmacology
• if too concentrated, fatal paralytic ileus,
  abdomen dilated, do badly and die
• correct concentration is 36 mg/ml or less

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Permanent iv-access
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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

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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

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Complete inhalation set
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Induction chamber
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Nose cones with exhaust
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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
www.agnthos.se
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Inhalation

• 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

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Isoflurane

• Combines reasonable research anaesthesia and good
  guidance
• more expensive than halothane
• requires own vaporizer
• no mortal concentrations at room temperature

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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

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CO2

• 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

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Postoperative care

• temperature
• infrared light bulb
• insulation
• fluid
• ip or sc as boluses
• air humidification
• additional oxygen
• carbogen flow to chamber

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Insulated, recovering rat
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Recovery chamber
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Eyes stay open and.dry
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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

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Pain?

• 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

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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

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Why pain?

• It is a warning signal
• specific part of body will not be used
• Pain can also be harmful
• lack of use spasms -gt weakness, loss of muscle
  and permanent change
• Pain can result in lack of appetite and drinking

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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

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Pain assessment

• rodents are nocturnal
• animals should not notice
• infrared lamp during dark
• handling responses
• www.lal.org.uk/pdffiles/fleck.pdf

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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

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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 -gt recovery delayed

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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

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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

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Continues..

• 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

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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

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Pain behaviors..

• Falling
• temporary loss of balance
• falls to side or backwards
• Each of all three are counted as one

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Ideal analgesic

• Does not interfere the study
• No sedation
• Long duration
• Efficient analgesia

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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

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Methods

• systemic analgesia
• duration?
• local analgesia
• applied on the incision area

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Analgesics

• 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(RM) mg/kg sc or im / 3 h
• Morphine 2-5(RM) mg/kg sc /4 h

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Other

• Rat (R) and Mouse (M) doses
• many given per os / dissolve in water
• Carpofen 5(RM) 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

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Per os administration
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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

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How to use ?

• Small surgical procedures -gt single injection
  e.g. buprenorphine
• in major interventions continue 2-3 days
• Additionally place local analgesia into the
  incision
• Follow animals and verify efficacy

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Books

• 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.