Pain, anesthesia, surgery and euthanasia

Question 1

Examples of behavioral changes and other signs of discomfort, pain, suffering, or distress in a rat or a mouse

Answer 1

1. Face: Grimace expression (whiskers back, eyes closed, ears flattened back, bulcky nose and flattened cheeks)
2. Body posture: Hunched and scruffy
3. Lack of activity: No nest building
4. Urinating and defecates more (diarrhea)
5. Under grooming
6. Not active
7. Weight loss
8. Stereotype behavior including over grooming (cannot cope with the environment)

Question 2

Examples of signs of positive well-being in a rat or a mouse

How to avoid:

Answer 2

Nest-building
Social activity
Explorative
Grooms itself (good looking fur)

Avoiding pain and distress is essential – gentle handling of the animals, optimal environment, cage mates (single housed mice are stressed), avoid fighting, pain relief if necessary according to the experiment.

Question 3

Examples on principles of how pain, suffering and distress can be managed

Answer 3

Assessment protocol: Favorable to have as it guides you and the caretakes in what to look for. There should be a specific protocol for that specific experiment and species/strain of animal.

Subjective pain assessment score: pain yes or no: 1-10 points.

Objective pain assessment: echtogram, grimace scale, activity: yes/no, nest-building: yes/no
Optimize the welfare of the animal by optimize the surroundings and gentle handling of the animal included train the animal for the procedures.

Question 4

What is a humane endpoint?

Answer 4

A humane endpoint is stated within the application for the license. At what point (clinical signs including weight loss) does the animal have to be euthanized - excluded from the experiment? Even though the experiment is not finished yet and you haven’t collected all your needed data (experimental endpoint).

Question 5

Examples of humane endpoints (mice)

Answer 5

Criteria’s must be relatively objective – so everyone handling the animals assess them evenly, it could be:
- Weight loss (procentage of weight/BCS)
- Heart rate/respiration rate – hard to observe
- Hunched posture and scruffy fur = bad welfare
- No nest building/non-active animals
- Grimace scale (mouse/rat)
- Diarrhea
- Coughing
- Behavior: echtograms

Question 6

Model specific humane endpoints (tumor/artheritis)

Answer 6

Model specific: tumor size, wounds yes/no, altered gait due to tumor: yes/no

Question 7

Humane endpoint scoring sheets

Answer 7

You must know the normal behavior, anatomy and physiology of the species as well as signs of pain in the specific species.
If the animals have been genetically modified you must take into consideration that there is a risk that it won’t act like a normal animal.
- General condition (mice): explorative? Nest building? Eating? Drinking? Fur (nice or scruffy)?
- Posture: hunched / abnormal / normal
- Movement: abnormal (paralyzed/hypermetric) / normal
- Weight loss? 5 % might be too much in normal mice, and 20% might be the humane-endpoint for tumor-model mice

Question 8

Humane endpoints - options for refinement - is it possible to finish at an earlier endpoint?

Answer 8

Refinement: (reduction of stress, fear and distress) if you already know that the drug you are testing will affect the animal in a specific way and that the animal only will get worse over time, then the animal must be euthanized when seeing the first small clinical signs

Question 9

EU Directive Severity classification: cumulative severity/non-recovery/mild/moderate/severe

Answer 9

The license for the experiment has to describe the severity classification, does the animal suffer, feel pain, distress or lasting harm? Intensity, duration, frequency and multiplicity.

Cumulative severity: a procedure is done over and over – how is the animal affected – it must be assessed worse than if the pocedure was done once; “moderate” to be exposed to pain over and over again despite the fact the pain is considered “mild”.

Non-recovery: procedures under general anesthesia, the animal will not recover consciousness
Mild:
- Short-term mild pain, suffering or distress
- No significant impairment of the well-being or general condition of the animal
- E.g. administration of anesthesia or short term restriction

Moderate:
- Short-term moderate pain, suffering or distress
- Long-lasting mild pain, suffering or distress, or
- Moderate impairment of the wellbeing or general condition
o E.g. Surgery under general anesthesia

Severe:
- Severe pain, suffering or distress or long-lasting moderate pain, suffering or distress
- Servere impairment of the wellbeing or general condition of the animal
o E.g. servere restriction of movement over a prolonged period (the period has to be defined by the competent authority)

Question 10

Euthanasia - principles

Answer 10

Euthanasia: a good death. No stress or fear and as quick as possible. It must be safe for the person performing it.
The death must be confirmed:
- No circulation of blood/respiration stops
- Destruction of brain
- Dislocation of the neck
- Complete bleed of (exsanguination)
- Rigor mortis/drop of temperature

Question 11

Euthanasia - legislation EU directive: examples on methods

Answer 11

Cervical dislocation: rodents (rats awake must be <150g)

Carbon dioxide (gradual fill): rodents (adult)

Medical euthanasia (overdose of anaesthesia: Pentobarbital)

Different methods for the different species!

Question 12

Euthanasia - why must someone competent in killing animals be available at all times?

Answer 12

A person competent in killing animals should always be available as anything can happen and the need for a quick death is needed. No animal should suffer unnecessary according to both EU- and Danish legislation. If the animal gets hurt you need to euthanize it immediately.

Question 13

Define sedation, local anaesthesia and general anaesthesia

Answer 13

Sedation: depression of CNS - only the consciousness is altered, no pain relief

local anaesthesia: a small area with analgesia - fx nerve block

general anaesthesia: loss of consiousness and feeling no pain, preferred with muscle relaxation

Question 14

Explain the triad of anaesthesia

Answer 14

1. Loss of consciousness
2. Analgesia
3. Muscle relaxation

Only possible with a combination of drugs = balanced anaesthesia.

Question 15

Explain balanced anaesthesia

Answer 15

The mixture of drugs providing you with an unconscious animal, that feels no pain and with relaxed muscles throughout the entire procedure. Goes down well and recovers fast is optimal.
Advantage: You need less of each drug used and thereby avoids some of the adverse effects.

The use of multiple drugs:
o pre-operative: sedativa and opiods often used in large animals to reduce stress and fear (not used for rodents – they go down well with only the anasthesia)
o Anaesthesia: inhalation or injection (hard to handle the dept)
o intra-operative analgesia or supplementing anaesthesia
o post-operative: analgesia

Question 16

Discuss the pre-anaesthetic considerations necessary for a successful anaesthetic procedure:
acclimatization and evaluation of the animal

Answer 16

Acclimatization for 1-2 weeks –> the animal return to normal physiological parameters

Evaluation of the animal: reduce mortality, faster recovery, less variation in results.

Healthy (sick animals have increased risk of dying under anaesthesia).

Known health status (not subclinical ill)

Not stressed?

Fasting? Not in rodents (cannot vomit) – only large animals.

Question 17

Discuss the pre-anaesthetic considerations necessary for a successful anaesthetic procedure:
Selection of agents (mind the experiment)

Answer 17

The drugs chosen for anesthesia should be something you have experience with, or you colleagues can advise you in. it should be suitable for the species of the animal and the duration of effect must match the procedure – use literature and your experienced colleagues! Remember to use proper analgesia before, under, during and after the procedure being done!

The drugs influence on the parameters you wish to measure should be limited to minimum even if it gives you a less efficient anaesthesia.

Question 18

The advantage of using pre-anaesthetica? (large animals, not rodents)

Answer 18

Pre-anesthesia is not relevant in rodents but used for large animals – often sedatives to reduce fear and stress. Gives you a smother induction of the anasthesia and reduced the amount of drugs needed.

Question 19

Monitor anaestetic level: short-term

Answer 19

• Righting reflex
• Withdrawal reflex
• Body temperature
• Respiration
• Heart rate (circulation)

Large animals: palpebral reflex, corneal reflex

The swallowing reflex might be present in well anesthetized animals (50% of rodents.).
Simple monitoring as a pulseoximeter and rectal temperature

Question 20

Monitor anaesthetic leve: long-term

Answer 20

• Righting reflex
• Withdrawal reflex
• Body temperature
• Respiration
• Heart rate (circulation)

More complicated monitoring – ECG, direct arterial blood pressure (large animals) tidal volume/gas concentration (intubated on a respirator)

Question 21

Maintenance of anaesthesia: two options incl. advantages and disadvantages

Answer 21

Injection (hard to control the dept of anaesthesia, not as fast as inhalation). Very easy to use, minimum demands for equipment. Cheap!

Inhalation: fast induction and can easily be controlled, recover fast. Need of special equipment and a specific ventilated room (gasses) might need a hand for monitoring the anasthesia

Question 22

Describe what problems that might arise during anaesthesia and discuss what actions to be taken to prevent or counteract these.

Answer 22

Animal is moving: more anaesthesia/analgesia

Low body temperature: provide heat – heating plate/wrap in bubble plast, heated saline (SC, IV or IP for rodents)

Cyanosis: provide oxygen

Cardiac arrest: stop operating, adrenaline and CPR

Question 23

Describe and discuss methods of optimising post anaesthetic recovery to ensure a smooth and rapid recovery from anaesthesia.

Answer 23

• Secure maintenance of body temperature - heating pad/SC/IV or IP heated saline fluid.
• Provide sufficient analgesia pre, intra and post anaesthesia.
• Rodents must be touched as little as possible
• Be silent
• Provide rodents with recovery gel or ensure that the water nipples are long enough.
• Provide oxygen as long as possible

Large animals (pigs and dogs) react well with being petted and cared for while recovering – rodents not at all!

Question 24

Describe and discuss good working practices with regard to use, storage and disposal of anaesthetic and analgesic agents

Answer 24

The drugs must be held in a locked cabinet, you must record the uses of the drug and at any times know how much of the drug is used and how much you have left.
The drug and the syringe/needles used must be disposed according to national legislation if not needed anymore or the date is exceed.

Avoid spilling drugs when handling them – do not injure yourself with needles!

Question 25

Discuss strategies for successful perioperative pain management, and difficulties that may arise in connection to this

Answer 25

You must ensure that the animal is not suffering – it is only allowed for the animal to feel pain (suffer) when it is approved in the license for the experiment.

Analgesia is most effective if given before the nerves are activated = before the animal feels pain.
You must understand how the analgesia might conflict with the experimental data needed - chose the drug with the best analgesic effect with minimal impairment of your experimental data (literature!).
- Most opioids suppress the immune system - buprenorphine to a lesser extent
- NSAIDs can have an unwanted effect on bone healing – studies are not conclusive

It can be difficult to observe the heart rate and the respiration rate in rodents – but it might be the only indicators of pain. – use clear covers while operating.

Question 26

The relevance of pre-operative assessment

Answer 26

Assessment: is the animal healthy? No sub-clinical infections (rely on health reports) and proper acclimatized (1-2 weeks for rodents)

Healthy animals = better research quality!
1. reduces mortality during anaesthesia
2. faster recovery
3. less variation in results

Question 27

Halsteads principles - minimizing trauma and increases healing (good surgical practice)

Answer 27

Halsteds principles:
- Aseptic procedures
- Gentle handling of tissue
- Hemostasis
- Avoid dead space
- Approximation of tissue without tension = correct suture pattern
- Avoid foreign materials

An unexperienced surgeon will cause more damage to the tissue than an experienced surgeon = higher risk of complications. The surgeon must always be prepared and know the procedure!

Instruments must be cleaned and sterilized, keep your dirty instruments separated from the clean instruments.

Question 27

Good surgical practice including the competences of personnel

Answer 27

Plan the procedure in depth, prepare and train including the assistants.

Work aseptic
Proper assessment of the animal; pre- inter and post surgery.

Know your limits and seek guidance and help from experienced colleagues.

The animal must be induced in a silent, darkened room with a minimum of stress.

The recovery site should be separated from the operation site being darkened, silent and heated (rodent) with appropriate bedding.

The animal must be treated with analgesia, pre-peri- and post surgery. The only time your are not to relive the pain is if the pain is measured for the experimental data collection.

Question 28

Give examples of commonly used surgical instruments, suture materials and needles and how they are applied

Answer 28

Hemostat – pean – used for hemostasis

Material scissor – used for cutting suture and other materials

Needle holder

Tissue scissor – used for cutting tissue

Scalpel – cutting tissue

Suture material:
- synthetic such as vicryl - non-ansorbable monofilament outside and absorbable polyfilament inside.

Suture pattern: suitable for the tissue and tension

Correct needle: cutting/blunt - curved/straight ect.

Question 29

Discuss the principles of post-surgical care and monitoring, and describe common post-surgical complications and their causes

Answer 29

The animal recovering from surgery must be relieved from pain (analgesia provided pre-inter and post surgery).
The rodents prefer a silent, warm and darkened place to recover with no human contact (stressful)
The companion animals prefer tender loving care while recovering. In a warm, silent place.
The animal must be closely observed for any signs of pain (grimace scale, body posture, activity, attention to operation wound, water and food intake, urination (dehydration) and defecating). The animal must be supplied with sufficient fluid.

Complications:
- Infections and thereby prolonged recovery are common if not aseptic or an unexperienced surgeon.
- Dehydration – not sufficient supplied with fluid under surgery and not supplied post surgery
- Respiratory depression – anesthesia affects the respiration and the animal is not fully breathing by itself – supply with oxygen or even intubation
- Hypothermia – the temperature must be measured frequently. Adult: 25-30 and neonatal: 35-37 (incubator)
- Eye damage due to wrong bedding (sawdust is bad for the rats eyes and nose) and no eye protection under surgery