Anaesthesia & analgesia of laboratory animalsTimo NevalainenUniversity of Eastern Finland

TerminologyAnaesthesia = without sensationan = without, aestos = sensationAnalgesia = without painan = without, algios = painEuthanasia = good death eu = good, thanatos = death

How anaesthesia is chosen ?Traditionlook articles of your disciplineAsk colleaguesend up to traditionRational way ?

Anaesthesia, how ?Choice of anaestheticminimal interference of studyimmobilisation and no/less painnature of the procedureduration of the procedureHumane handling of animals and safety of personnel are important

Problems with laboratory animalsGroup anaesthesiaLarge species, strain etc. differencesFollow-up difficultiesAnaesthesia poorly knownClinical and research anaesthesia are not used properlyPostoperative care does not work

Pros and cons of anaesthesia Prosno painno muscle reflexesno muscle tensionClinical anaesthesia Conschanges in the bodyphysiological status differentresponses may be differentResearch anaesthesia

Anaesthesia vs. CNS-mediated reflexesDepresses leastdepresses mostalphachloralosethiobarbiturate + N2Ocyclopropanebarbituratesetherhalothanechloroform

Inspection before anaesthesiaCheck animals before startingDo not anaesthetise sick animals, they are unsuitable for experiments anywayPay attention to symptoms of infections

How to fast before anaesthesia ?Take away food - not waterthe smaller species, the faster metabolism, the shorter fastingno fasting for mouseabdominal procedures in rat, fast for 6 hguinea-pig 6 hrabbit 6 h

Dangers of vomitingHorse vomits = rupture of stomachruminants can drop ruminating ballscarnivores vomit easilyamong rodents guinea-pig vomits easilycan drain into trachea and cause aspiration pneumonia

Pre-anaesthetic hydrationGive animal balanced electrolyte fluid to drink couple of days beforeyields better water balancecontinue a few days after anaesthesia (and procedure)

Pre-medication atropineNo saliva, no vagal reflexes, less gut motilityDose: rodents 0.05 mg/kg, rabbit 1-3 mg/kg about 30 min beforeif autonomic nervous system is involved, atropine may be contraindicated

Sedative pre-medicationDecrease dose of anaesthetic by 20-50 %better handling may be needed eg for iv injection if procedure is not painful, but immobilisation is neededsome compounds dilate vessels - easier to see them

Ensuring oxygenationRespiratory passages openposture, atropineadditional oxygentubing directly into mouth less dead spaceintubation

ECG-recording

Pulse oxymeter

Maintaining normal temp

Control of anaesthesiaInhalation is well controllediv-bolus anaesthesia - you give half of calculated dose, the rest to effectinfusion anaesthesia also well controlledim, ip ja sc administration: you give calculated bolus - response may be variable

Inhalation - open mask

IntubationMouse - tracheostomyrat - tube outer diameter = 1-1.5 mm, length 2 cm attached snugly inside wider tuberat placed on back, fixed by maxilla incisors, tongue pulled outlarynx visible, becomes easier with high intensity light directed to neck

Intubation

Rabbit intubation difficultlaryngospasm unless not deep enoughsmall place, otoscope / pediatric laryngoscopetube outer diameter = 3 - 3.5 mm, no cuffRabbit: guided or blind intubation

Mouse intubation video

Air movement ?

Wet - loosing heat

Skin disinfection

Assessment of anaesthetic depth Mouserespiratory rate, corneatail pinch and pedal reflexpedal bestRatrespiratory rate, tail pinchpedal reflex and ear pinchear pinch best

Tail pinch

Pedal reflex

Anaesthetic depth

Rabbitlight surgical - pedal reflexmedium depth - palpebral reflex & ear pinch corneal reflex - dangerously deep

Ear pinch

Hypnorm & midazolamclinical anaesthesia, reasonable safety marginNOT to be given ip, liver metabolism weakens effectcontains fentanyl = controlled substancerecovery is speeded by nalorphinRat: Combination 0.15 - 0.2 ml / 100 g scMouse: Combination 0.10 - 0.15 ml / 20 g sc

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 minReversal: Nalorphine 1 mg / kg iv, im, ip

Medetomidine & ketamineRatMedetomidine 0.4 mg / kg + ketamine 60 mg / kg ip, sc. MouseMedetomidine 1 mg / kg + ketamine 75 mg / kg. Mixed and diluted to yield 0.1 - 0.2 ml / mouseAntagonist Atipamezole 1 mg /kg (rat) im, ip, sc or 0.5 mg / kg (mouse) ip, sc.

ChloralhydrateUsed to be common in neuropharmacologyif too concentrated, fatal paralytic ileus, abdomen dilated, do badly and diecorrect concentration is 36 mg/ml or less

Permanent iv-access

Barbiturate anaesthesiaSafety margin in rabbits narrowmay lead to 20-40 % mortalityif used - only for terminal proceduresMouse - the same situationcan combine with e.g. ethanolthere are better combinations

Complete inhalation set

Induction chamber

THE UNIVENTOR 400 ANAESTHESIA UNITdesigned to control the mixture of anesthetic and air with the precision required to successfully operate on animals weighing from 20-500 gramswww.agnthos.se

Inhalationhalothanecommon inhalation compounddoes not evaporate concentrations with mortality at room temperatureliver necrosischeapest of proper inhalation compoundsgood clinical anaesthesia with guidance

IsofluraneCombines reasonable research anaesthesia and good guidancemore expensive than halothanerequires own vaporizerno mortal concentrations at room temperature

No ether, neither chloroformEvaporate to mortal concentrations at room temperatureether explodes, and carcasses in cooler of freezer smell a long timechloroform is liver toxic and suspected carcinogen

CO2Controversial1-2 min procedures, no longerfor rats and miceincubation box with animals, tube CO2 in at a rate 0.6 x box volume l/minrighting reflex disappears, move to mouth cone with 50 % CO2 and 50 % O2

Postoperative caretemperatureinfrared light bulbinsulationfluid ip or sc as bolusesair humidificationadditional oxygen carbogen flow to chamber

Insulated, recovering rat

Recovery chamber

Eyes stay open and.dry

Strategy for research anaesthesiaSimple is best - avoid polypharmacyEffect on your studyPrefer inhalationstable anaesthesiacan measure inhalant concentration from expired air = best description of anaesthesia

Pain?IASP definition: An unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage. Pain is always subjective ()IASP 1979

Are animals capable of feeling pain?Criteria:Similar anatomical and physiological mechanisms to those involved in pain perception in manDisplay of behavioural response to painResponse inhibited by analgesics drugs(Smith and Boyd, Lives in the Balance, 1991)

Animal painmay not be identical to humansit is assumed that intensity and duration of pain is variable in specific tissue damage between speciesfast recovery leads to underestimation of pain and failure to give analgesic

Why pain?It is a warning signalspecific part of body will not be usedPain can also be harmfullack of use & spasms -> weakness, loss of muscle and permanent changePain can result in lack of appetite and drinking

Pain assessmentdifficult in humans even though we understand what they sayanimal approachesanthropomorphic waybased on clinical findingsactivity, grooming, immobility, vocalization, posture, aggressiveness

Pain assessmentrodents are nocturnalanimals should not notice infrared lamp during darkhandling responses

http://www.ahwla.org.uk/index.html

Pain assessmentPhysiological parametersHR, BP, BT Biological markersCathecholaminesCorticosteroids Behavioural and clinical signsAnalgesiometrySubjective assessmentObjective assessment

Signs of painSpecies and procedure specificScoring system is a necessityanalgesic treatmentverification of analgesia efficacypostoperative analgesia the most common formhumans with neoplasia and infections receive analgesiafear for interference in animal studies

Difficulty of assessmentAnimal shows no sign of painpain will not be treatedComparison to human situationbetter to give a single doserepeated dose may be problematice.g. appetite may be lost -> recovery delayed

Pain scoring requiresknowledge ofspecies specific behaviorbehavior before the procedurepalpation of the tissue & response evaluation of sore area (e.g. leg)knowledge on efficient analgesics doses, and possible behavioral consequences

Ideal analgesicDoes not interfere the studyNo sedation Long durationEfficient analgesia

Analgesia; contraindications Analgesia does NOT replacegood surgical techniquegood peri- and postoperative careAnalgesia should not interfere with the study or interpretation of the results

Choose appropriate analgesicOpioidsWeak: Codeine, dextropropoxiphenStrong: Morphine, fentanyl, buprenorphineNSAIDsCarprofen, ketoprofen, meloxicamLocal anaesthesiaCombinations

AnalgesicsRat (R) and Mouse (M)Opioids - look for durationBuprenorphine 0.05(R), 0.1(M) mg/kg sc / 6-12 hButorphanol 2.0(R) mg /kg sc / 1-2 hPetidine10-20(R&M) mg/kg sc or im / 3 hMorphine 2-5(R&M) mg/kg sc /4 h

OtherRat (R) and Mouse (M) dosesmany given per os / dissolve in waterCarpofen 5(R&M) mg/kg sc or per osR 12-24hFluniksine 2.0(R) mg/kg sc /1-2hKetoprofeine (R) 5 mg/kg im/12-24h

Per os administration

Analgesia - advantagesFaster recoveryFaster return of appetiteNo weight lossWhich is better:effect of pain and procedure or only effect of procedureEthically right

How to use ?Small surgical procedures -> single injection e.g. buprenorphinein major interventions continue 2-3 days Additionally place local analgesia into the incisionFollow animals and verify efficacy

Pain assessment after laparatomy in ratsStage 1: Visual Analogue Score (VAS)assess pain experienced by the rat with a mark on a 10 cm linethere are 4 clips to assess

Continues..Stage 2: Instructions become familiar with behaviors typical to paindo not count at this pointOnly three behaviors needs to be recognizedorder haphazard like in real life

Pain behaviors 1Twitchesusually when the rat restsmost common on back, fur coat movementalso on the headmost common behaviorBack archingfeet extended, belly goes up, arching backoften just prior to walking

Pain behaviors..Fallingtemporary loss of balancefalls to side or backwardsEach of all three are counted as one

BooksP.Flecknell. Laboratory Animal Anaesthesia. Academic Press, 1996H.B. Waynforth & P.A. Flecknell: Experimental and Surgical Technique in the Rat. Academic Press 1992D.H. Kohn, S.K. Wixson, W.J. White, G.J. Benson. Anesthesia and Analgesia in Laboratory Animals. Academic Press 1997.

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