5.6.

Anaesthesia and Analgesia

to minimise pain, suffering,
distress or lasting harm

Dr Katsimpoulas Michalis DVM, SRS, PhD

Staff Research Scientist
Assistant Professor Level
Center of Clinical, Experimental Surgery and Translational Research
Biomedical Research Foundation of the Academy of Athens
 Historical Overview

• 3rd Century BC, Alexandrian Physicians Herophilus and Erisistratus

examined functional differences between humans and animals.
 Historical Overview

• Belgian Andreas
Vesalius (1514-64) in
Padua, Italy,
illustrated public
lectures on anatomy
vivisecting executed
humans
 Historical Overview

• Animals have no
capacity for reasoning,
thus no perception of
pain

1596-1650, Philosopher-Mathematician
 Historical Overview

• Primatt and Bentham (1789)

replaced the point of whether an
animal had a soul or not to a new
criterion: the capacity of animals
to experience pain and suffering
• William Russell and Rex Burch: The
Principles of Humane Experimental
Technique (1959) pioneered the
notion of the “3Rs” (Replacement,
Refinement, Reduction)
A physiological demonstration with vivisection of a dog. Oil painting by Emile-Edouard
Mouchy, 1832. From the Wellcome Library, London.
 What are The 3r’s
 Replacement
Methods that avoid or replace the use of animals
 Reduction
Methods that minimize the number of animals used
per experiment or study
 Refinement
Methods that minimize the pain, suffering, distress or
lasting harm that may be experienced by the
animals.
Anesthesia is derived from the Greek anaisthesis, meaning “loss of sensation”
 The goals of anesthesia

 eliminate distress
 eliminate the perception of distress/ pain to certain
extend during experimental procedures
 immobilize the animal during experimental procedures

 These goals must be accomplished without posing a

serious threat to the life of the animal.
 why must be done properly ?

 Ethical Reasons (3R’s)

 High quality data

 Appropriate anesthesia and analgesia procedures

must be established to ensure that clinical,
welfare (humane end points), and research
requirements are met.
 animal considerations

 Age  Body weight

 Sex  Health status
 Genotype  Pregnancy
 Light cycle  Concurrent medication
 Strain differences  Previous anesthesia

1. Mice < 8 weeks old metabolize anesthetics less efficiently compare to adults
2. Mice > 18 months old we have the same problem because to subclinical pathologies related with cardiac, renal and hepatic function
3. Gender influences the pharmacokinetics, metabolism and other physiological parameters. These observed due to differences in sexual
hormones, corticosteroids, stage of estrus and in hepatic enzymes
4. Genetically modified rodents may have unexpected variability in their response to anesthetic compared to background strains
5. Rodents are nocturnal animals. cardiovascular function, blood constituents, gastrointestinal function, and endocrinology also follow a daily
cycle
6. Obese mice: alteration in biodistribution of lipophilic agents and a high incidence of hepatic dysfunctions, hence they have high anesthetic
risk
7. Cachectic mice: low plasma protein binding and perhaps subclinical pathologies related with cardiac, renal and hepatic function
 Consequences of small body size

 There is a limitation to on the volume that can

be administered

Specie Oral SC IP IM IV(bolus) IV(infusion)

s
Mouse 0,25 Scruff 2-3ml 2-3ml Quadriceps or Lateral tail Max
ml (<20G) (<21G) caudal thigh vein 25ml/kg
0,05ml (<23G) 0,2ml (<25G)
Rat 3- Scuff or 5-10ml Quadriceps or Lateral tail Max
5ml back (<21G) caudal thigh vein 20ml/kg
(Gaertner, Hallman et al. 2008)
5-10ml
(<20G)
0,3ml (<21G) 0,5ml (<23G)
 Consequences of small
body size
 High metabolic rates
 High oxygen consumption
 Small total volume of blood
 Small airways
 Project considerations

 Anesthesia must have as little as possible or no

influence on the quality of the results.
 It is important to review literature carefully and/or
contact a veterinarian for more information
 Do not rely in previously publications.
 Appropriate anesthetic agent should be used for
each individual project
PREPARATION FOR
ANESTHESIA
 Acclimatization

 Animals that have been recently shipped should be housed

for 1-2 weeks in order to acclimatized to the new
environment.
 Daily observation to check that they are in good health.
 Preanesthetic patient
evaluation
Healthy Diseased

Photos: www.humane-
endpoints.info
 Preoperative fasting
no yes
 Is unnecessary to  In gastrointestinal
rodents because they tract surgery
cannot vomit  Imaging studies
 Prolong fasting cause
hypoglycemia

Animals should always have free access to water, even

shortly before anesthesia
TYPES OF
ANESTHESIA
Local anesthesia General anesthesia
 Regional and reversible  Complete
elimination of pain unconsciousness
 Used in larger species  Rat, Mice
 EMLA cream takes 30-45
min to be effective. It is
useful for venipuncture.
Local Anesthetics
 Local Anesthetics

 The “-caine” family: Lidocaine, bupivicaine,

mepivicaine, proparicaine, tetracaine, etc.
 Local Anesthetics
 Prevent central sensitization since the nociceptive
signal is blocked
 Classified by duration of action
– Lidocaine is short acting with a rapid on-set
– Bupivicaine is long acting with a slow on-set
– Duration of action can be extended by adding a
vasoconstrictor like epinephrine
 Local Anesthetics
 “Splash”
– Can apply to exposed tissues prior to closure and
nerves prior to transection during amputations
 Infiltration
– Multiple intradermal or subcutaneous injections of
local anesthetic along proposed incision line-rat
GENERAL ANESTHESIA
 Stages of Anesthesia
 Stage 1 (Induction, aka voluntary excitement).
 Stage 2 (delirium, involuntary excitement).
 Stage 3 General Anesthesia
 Plane 1—Light anesthesia. Most reflexes are still present.
 Plane 2 – Medium anesthesia. Most reflexes are absent.
 Plane 3—Deep anesthesia.
 Plane 4—Too Deep.
 Stage 4 Irreversible Anesthesia
 Anesthetic methods for routine use in rodents

 Injectable anesthetics
 Inhalant anesthetics
INJECTABLE ANESTHESIA
advantages disadvantages
 Multiple animals can  Once the anesthetic has
be anesthetized at the been administered the
same time dosage cannot be reduced
 Do not require the use  There is individual
of specialist variability
equipment  The best way to give these
agents is intravenous - iv.
Routes of administering anesthetics
 Subcutaneous (SC)
– Can be used, but the onset time of anesthesia is slow
– Common sites: usually made into the loose skin over
the neck and upper back.
 Intramuscular (IM)
– Difficult in rodents because of the smaller muscle mass
that is available to inject into. Avoid injecting into or
around the sciatic nerve
– Common sites: hind limb muscles
 The Most commonly used
Injectable agents
 Barbiturates
– Pentobarbital
 Dissociative Anesthetics
– Ketamine (in combination)
 Neuroleptanalgesics
– Fentanyl/Fluanisone
 Other Hypnotics
– Xylazin (a-2 adrenergic agonist)
– Etomidate and Metomidate (a-2 adrenergic agonist)
– Diazepam and midazolam (Benzodiazepines)
– Propofol
 Pentobarbital
 Pentobarbital has been the most widely used
laboratory animal anesthetic.
 Provides surgical plane of anesthesia following
IV or IP administration
 Strain effects sleeping time
 Pentobarbital
 Have a narrow margin of safety
 In rodents provides minimal analgesia
 this agent will decline in the future

Specie Dosage (mg/kg) and Duration of Comments

s route of surgical
administration anesthesia (min)
Mouse 40 – 70 i.p. 20 – 40 Cardiorespiratory depression,
Rat 30 – 50 i.p. 15-60 hypotension, long duration 120-180
min for full recovery
 Ketamine
Alone Produces:
 a state of catalepsy,
 superficial analgesia
 immobility in most animals
with a marked increase in
skeletal muscle tone.
 light respiratory depression
while preserving
cardiovascular function
In combination
 Produces:
 muscle relaxation
 prolonged duration of
anesthesia
 When is given sc or im,
may also cause localized
tissue necrosis
 Ketamine + xylazine
 Is still the most widely used ketamine combination
 Provides good immobilization with some degree of analgesia
 Large variability of the recommended dosages
 Side effects
– Bradycardia and increased peripheral vascular resistance
– May lead to respiratory acidosis
– Hypothermia (↓ 4C° over 60 min of anesthesia)
– Acute reversible cataracts have been documented in mice and
rats.
– Development of corneal lesions in rats
 Ketamine + medetomidine

 Is about 10 time more powerful than xylazine and that

exhibits high selectivity for a-2 receptors
 Its side effects are fewer and not as pronounced.
 May cause hyperglycemia and polyuria similar to
xylazine
 Female mice appear to be more resistant to the
effects of this combination than male mice
 Alpha-2 antagonist

 The effects of both xylazine and medetomidine can be

reversed by atipamezol.
 1 mg/kg i.p. or s.c. for mice and rat
 Ketamine + diazepam or midazolam
 Midazolam has shorter onset and duration compared with
diazepam.
 Recommended for old animals and diseased patients
(cardiovascular, metabolic)
 Provides only light anesthesia
 Can be antagonized in rodents by flumazenil (10 mg/kg i.p.)
 recommended for old animals and diseased patients including cardiovascular and metabolically
compromised patients
Species Dosage (mg/kg) and route of Duration of anesthesia Comments
administration (min)
Mouse 100 K/5 D or M 20 – 30 Immobilization
Rat 75 K/5 D or M 20 - 30 Immobilization
 Fentanyl/Fluanisone
 Produce a state of deep sedation with profound analgesia
sufficient to perform short painful procedures
 They can be reversed by naloxone (0,01 – 0,05 mg/kg i.v.,
i.m., i.p.)
 The advantage of this combination being that at any time
each or all of the agents can be reversed
 The main side effects related to the use of opioids are
respiratory depression and bradycardia (Buprenorphine can be
administered to reverse the respiratory depressant)
 Propofol
 Is administered IV
 Produces rapid induction of a short period of anesthesia.
 Side effects
– Myocardial depression
– Bolus injection resulting in apnea and hypotension
– The injection may be painful in rodents.
 It is recommended to be used within 6h of opening a vial to
avoid the potential growth of microorganisms
Species Dosage (mg/kg) and route of Duration of Comments
administration anesthesia
(min)
Rat 7,5-10 IV followed by 44 to 55 mg/kg/hr 5 – 10 (bolus) 10-20min
recovery
INHALATION ANESTHESIA
 Advantages of inhalation anesthesia
 They are simple to administer
 Induction of anesthesia is usually smooth and
rapid
 Predictable and rapid control of anesthetic depth
and duration
 Rapid recovery (10 – 15 min)
 Minimal influence on research data
 They are not controlled substances
 Disadvantages of inhalation anesthesia
 Induction must be closely monitored
 Specialized personnel training may be necessary
 Specialized equipment is needed
 Costs associated with training and equipment
 Only one animal at a time
 Risk of occupational exposure to waste anesthetic gas
 Requires compressed gas, oxygen source and vacuum
 Inhalational agents

 Isoflurane
 Sevoflurane
 Desflurane
 Halothan
 Methoxyflurane
 Enflurane
 Ether (Discontinued)
 Isoflurane
Advantages
 Rapid induction and recovery
 Comparatively little effect on
cerebral blood flow and
cerebrospinal fluid pressure
 Minimal effects on coronary blood
flow and cardiac output; protective
effect in models of ischemic
cardiac disease
 Minimally metabolized by the liver
 Good muscle relaxation
 It is non-irritant, non- explosive and
nonflammable
Cardioprotective effects can last for 2 weeks following the anesthetic episode
disadvantages
 Expensive
 Pungent odor (can cause animals
to hold breath during induction)
 Increased airway secretions and
reflexes
 Requires vigilant monitoring, since
the depth of anesthesia may
change rapidly
 Dose-related cardiovascular and
respiratory depression
 sevoflurane
 Can provide an even faster anesthetic induction
and recovery compared with isoflurane
 The depth of anesthesia can be altered very
easily and rapidly
 It is nonexplosive and non-flammable
 Is much less pungent than other agents
 It can induce respiratory depression and
hypotension in a dose-dependent manner
 Its high cost does limit the use in laboratory
animals
Anesthetic Concentration for Concentration for
induction of maintenance (%)
anesthesia (%)
Isoflurane 5 1,5 – 3
Sevoflurane 8 3,5 – 4
Desflurane 18 11
Equipment

INHALANT ANESTHESIA
 Bell jar
 The use of a “bell jar” for short-duration anesthesia
continues to be popular because the required
equipment is inexpensive and readily available
 Bell jar
 Disadvantages
 Contact with the liquid anesthetic
 An unmeasured and uncontrolled
concentration of the anesthetic
 The possibility of accidental overdose
 Release of anesthetic to the room air
INTUBATION
 materials
 Light source
 Anatomic forceps or two cotton swabs
 Lidocaine spray
 Intravenous catheter Mouse: 20G, Rat: 16-18G
 Tape
 Anesthetic
 Tilt bed
 Elastic band
Mouse intubation
 Rat intubation
Rat intubation

Intubation in the rat. (A) This photograph of a laryngoscope with a light source incident on the proximal end illustrates how light is transmitted to
the distal surfaces. (B) Image of laryngeal opening of a rat showing the epiglottis (red arrowhead), Arytenoid cartilages (black arrows), and
caudal margin of the soft palate (black arrowhead). Visual appearance is similar in the mouse. (C) An anesthetized rat positioned and restrained
on inclined plane. (D) The laryngoscope is positioned in the oral cavity to provide visualization of the larynx. The tongue is grasped against the
shaft of the laryngoscope. The stylet and tracheal tube are shown before being inserted into the oral cavity. Note the relative position of stylet
within the tracheal tube. (Reprinted with permission from AALAS..)
Etiology of rodent intubation
complications
ANESTHETIC DEPTH
 Pedal withdrawal reflex: if it’s positive anesthesia
needs to be deepened before surgery is begun.
 Monitoring the rate and quality of the pulse, if the pulse
is readily palpable.
 Monitoring the heart rate and blood pressure if
applicable.
 Post Anesthetic Recovery (21.16)

Post-operative care = good anesthetic practice.

Failure = complicate recovery = inhumane.
Poor post-operative care = prolonged metabolic = death.
 Post Anesthetic Recovery (21.16)

The recovery area environment must be appropriate for the species. The
continued provision of supplemental warmth, fluid nutritional support and
nursing care is often necessary.

Temperature 27– 30 ° C for adult animals and 35– 37 ° C for neonates. Once
recovered from anesthesia, temperature can be reduced to 25 ° C for adults,
but maintained at 35 ° C for neonates.
 Post Anesthetic Recovery (21.16)

Recovery in normal cages.

Placed either in a recovery room or inside an incubator.

Sawdust or wood shavings as bedding is forbidden, as it sticks to the

animal’s eyes, nose and mouth

Synthetic bedding with a texture similar to sheepskin (fleece) has proven

particularly useful
ANALGESIA
 Circumstances anesthesia or analgesia necessary to
minimize pain, suffering, distress or lasting harm (5.6)

 Animals feel pain just like us

 Unethical not to address pain
 Public concern
 Many anesthetics have no analgesic effect
 Pain results in poor anesthetic recovery
 MYTHS

“Anesthetics mask symptoms”

“Patient will harm itself if there’s no pain”

“Pain is difficult to assess”

 In Reality
 Pain :
– Decreases cardiovascular function
– Decreases appetite
– Slows wound healing
– Decreases immune function
 Greater chance of infection
– Increases fear and anxiety
 In Reality
 Pain :
 Analgesic regimen should be determined by
assessing
 The intensity and duration of pain
Anesthetic agents with low analgesic effect (21.8)

• Anticholinergics (atropine)
• Phenothiazines
(acepromazine)
• Butyrophenones
• Oxybarbiturates
(pentobarbital sodium)
• Thiobarbiturates (thiopental
sodium)
• Propofol
• Etomidate
• Inhalation anesthetics
 So What Do I Need To Know?

 Definitions
 Types of pain
 Physiology of pain
 Analgesics and their action
 Pain assessment
 What is Pain?

• Accepted definition:
• An unpleasant sensory (noxious) and emotional
experience associated with actual or potential
tissue damage
 Definitions
• Analgesia – the absence, or decrease, of
pain in the presence of a stimulus that would
normally be painful
• Nociception – the reception, conduction, and
central nervous processing of nerve signals
resulting in the perception of pain
 Types of Pain
Physiological Pain Pathological Pain
• Protective mechanism • Tissue injury
• Causes avoidance • Inflammation
• Little to no tissue injury • Nerve damage
• Pain stops once the • Persists after the stimulus
stimulus is removed is removed
 Types of Pain
Acute Pain
• Occurs immediately after
a stimulus is received
• Severity can vary
• Responds well to
treatment
• Subsides once stimulus is
removed
Chronic Pain
• Persists well past initial
stimulus Severity can vary
• May or may not respond well
to treatment; may require a
“multi-modal” approach
 Physiology of Pain
Pain ≠ Nociception
What is the difference?
Pain is a product of higher brain center processing of signals
it has received.
Nociception refers to the peripheral and central nervous
systems processing information generated by stimulation
of nociceptors by noxious stimuli
Nociception can occur in the absence of pain.
 Physiology of Pain
There are four distinct processes involved in nociception
which can be modulated by analgesics:
– Transduction – translation of the noxious stimulus into
electrical activity at the peripheral nociceptor
– Transmission – the propagation of nerve impulses
through the nervous system
– Modulation – modification of nociceptive transmission
by inhibition of the spinal dorsal horn cells by
endorphins
– Perception – the final conscious subjective and
emotional experience of pain
Pain sensor nerve fiber spinal cord brain
 Actions of Analgesics on Pain
Processes

 Pre-emptive analgesia: giving analgesics prior to the noxious stimulus

(surgery)
 Multimodal or “balanced” analgesia: using a combination of analgesics
which will impact more than one portion of the nociceptive process
 Analgesics
 Divided into five main classes based on
mode of action
– Opioids
– Non-steroidal anti-inflammatory drugs
– Local anesthetics
– Alpha2-adrenoceptor agonists
– Miscellaneous drugs
 Opioids: Backbone of Analgesia

• Pure Agonists
• Morphine, oxymorphone, meperidine, hydromorphone, fentanyl
• Partial agonists, mixed agonist-antagonists
• Buprenorphine
• Butorphanol
• Pure Antagonists (reversal of agonists)
• Naloxone
• POTENTIAL ABUSE
 Opioids
 Agonists – include morphine and fentanyl
– Potent analgesics
– Can be reversed with naloxone
 Opioids
 Mixed agonist-antagonist – includes butorphanol
– Have agonist or partial agonist activity at one or more
opioid receptors and the ability to antagonize the
effects of a full agonist at one or more opioid receptor
– Less respiratory depression than full agonists
– Can be used post-operatively to reverse the narcosis
of fentanyl while still providing analgesia
 Opioids
 Partial Agonist – includes buprenorphine
– Buprenorphine has a prolonged duration of action
(relatively)
– ceiling effect
 Opioid Administration
 Systemic: IV, SQ, IM,
 Non-steroidal Anti-inflammatory
Drugs (NSAIDs)
 Weak organic acids with anti-inflammatory, analgesic, and
antipyretic properties
 Carprofen and meloxicam are COX-2 selective inhibitors
which have a reasonable margin of safety when used pre-
operatively
Non-Steroidal Anti-inflammatory
Drugs ( NSAIDS )
 Aspirin
 Carprofen - Rimadyl ®
 Meloxicam- Metacam ®
 Etodolac - Etogesic®
 Ketoprofen - Ketofen ®
 Phenylbutazone – “Bute”
 Flunixin - Banamine ®

 (Acetaminophen - Tylenol ®)
 NSAIDS
 Most have effective somatic (superficial) analgesic
effect
 Some have good visceral analgesic effect also
 All take 30-60 minutes to take effect, even if injected
 All have antiinflammatory properties
 Reduce fever
 Alpha2-Adrenergic Agonists
 Stimulation of the alpha2 – adrenoceptors
result in sedation, muscle relaxation, and
analgesia
 Can be reversed with alpha2-adrenergic
antagonists such as yohimbine and
atipamezole
 Includes xylazine, medetomidine,
detomidine and dexmedetomidine
 Xylazine: Good Things

 Moderate analgesia

 Potent sedative effect

 Good muscle relaxation

 Miscellaneous Analgesics
 Tramadol
– Synthetic opioid agonist

 Ketamine
– In rodents analgesia “is not enough” as a sole
agent
 Gabapentin
– Used to treat nerve pain
 Non-pharmacological methods

 Good nursing care

– Comfortable bedding
 Clean and dry
– Easy access to food, water
– Rotate recumbency
– Allow time to sleep
 Non-pharmacological Methods
 Apply cold to site (acute- 1st 24 hours)
– Decreases inflammation
 Apply heat to site (chronic)
 Massage
 Acupuncture/acupressure
 Complementary methods
 Preemptive Analgesia
 If the body doesn’t sense the pain during the
procedure, the pain will be easier to deal with
post-operatively

– A patient in surgical anesthesia is not aware of pain, but

the body is still responding sensitizes the nervous
system
Preemptive Analgesia Results In
 Marked decrease in amount of analgesic
medication needed post-operatively
 Increased patient comfort
 5.6. Anaesthesia or Analgesia to minimise pain,
suffering, distress or lasting harm

“If you are unable to justify aspects of your work to yourself, you will
have difficulty justifying them to others”

Vaugham Monamy