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Anaesthesia of laboratory animals

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Title: Anaesthesia of laboratory animals


1
Anaesthesia of laboratory animals
  • Timo Nevalainen
  • Universities of Kuopio
  • Finland

2
A more pharmacologic presentation www.uku.fi/tne
valai/Anesthesia.ppt
3
Terminology
  • Anaesthesia without sensation
  • an without, aestos sensation
  • Analgesia without pain
  • an without, algios pain
  • Euthanasia good death
  • eu good, thanatos death

4
How anaesthesia is chosen ?
  • Tradition
  • look articles of your discipline
  • Ask colleagues
  • end up to tradition
  • Rational way ?

5
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

6
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

7
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

8
Anaesthesia vs. CNS-mediated reflexes
  • alphachloralose
  • thiobarbiturate N2O
  • cyclopropane
  • barbiturates
  • ether
  • halothane
  • chloroform
  • Depresses least
  • depresses most

9
Inspection before anaesthesia
  • Check animals before starting
  • Do not anaesthetise sick animals, they are
    unsuitable for experiments anyway
  • Pay attention to symptoms of infections

10
Bedding Volatile Compounds / With Highest Sums
concentration microg/g
Vesel et al. 1966 Bedding volatile compounds
induce liver microsomal enzymes
11
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

12
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

13
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

14
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)

15
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

16
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

17
Ensuring oxygenation
  • Respiratory passages open
  • posture, atropine
  • additional oxygen
  • tubing directly into mouth
  • less dead space
  • intubation

18
Pulse oxymeter
19
Sensor around leg
20
Maintaining normal temp
21
ECG-recording
22
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

23
Inhalation - open mask
24
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

25
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

26
Mouse intubation video
27
Using otoscope
28
Air movement ?
29
Rodent respirator
30
Wet - loosing heat
31
Skin disinfection
32
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

33
Tail pinch
34
Pedal reflex
35
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

36
Ear pinch
37
Dose memos
  • www.uku.fi/tnevalai/ratmouseanes.html
  • Rabbit dose calculator
  • www.morfz.com/rx/drugcalc.html

38
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

39
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

40
Medetomidine fentanyl
  • Works reasonably well in rats
  • animals pale and urine drips
  • major advantage antagonist wakes them real
    fast

41
Chloralhydrate
  • Common in neuropharmacology
  • if too concentrated, fatal paralytic ileus,
    abdomen dilated, do badly and die
  • correct concentration is 36 mg/ml or less

42
Permanent iv-access
43
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

44
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

45
Complete inhalation set
46
Induction chamber
47
Nose cones with exhaust
48
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
49
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

50
Isoflurane
  • Combines reasonable research anaesthesia and good
    guidance
  • more expensive than halothane
  • requires own vaporizer
  • no mortal concentrations at room temperature

51
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

52
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

53
Postoperative care
  • temperature
  • infrared light bulb
  • insulation
  • fluid
  • ip or sc as boluses
  • air humidification
  • additional oxygen
  • carbogen flow to chamber

54
Insulated, recovering rat
55
Recovery chamber
56
Eyes stay open and.dry
57
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

58
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

59
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

60
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

61
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

62
Pain assessment
  • rodents are nocturnal
  • animals should not notice
  • infrared lamp during dark
  • handling responses
  • www.lal.org.uk/pdffiles/fleck.pdf

63
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

64
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

65
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

66
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

67
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

68
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

69
Pain behaviors..
  • Falling
  • temporary loss of balance
  • falls to side or backwards
  • Each of all three are counted as one

70
Ideal analgesic
  • Does not interfere the study
  • No sedation
  • Long duration
  • Efficient analgesia

71
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

72
Methods
  • systemic analgesia
  • duration?
  • local analgesia
  • applied on the incision area

73
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

74
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

75
Per os administration
76
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

77
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

78
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.
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