Microbiology 501

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Microbiology 501

• Alternatives
• Minimizing Pain and Distress
• Humane Euthanasia

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What are the alternatives?

• ..to the use of animals in research, education,
  and training??

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Alternatives to Use of Animals

• Rationale for considering alternatives
• Regulatory
• Ethical
• Economic
• Scientific

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Alternatives Search
Are there methods that can be used that will have
less or no impact on the animal without
compromising experimental integrity ?
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Ethics and Alternatives
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eplace
R
educe
R
R
efine
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Types of Alternatives

• Inanimate systems
• In vitro methods
• Non-traditional animals
• Advanced in vivo systems

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Replacement

• Absolute replacement - no use of animals used at
  all
• Relative replacement - humane killing of an
  animal to provide cells, organs and tissues for
  in vitro studies
• Uses fewer animals

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Absolute Replacement

• Vascular Access Training Models
• Forelimb
• Head-neck

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Absolute Replacement
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Relative Replacement
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Ascites Production of Monoclonal Antibodies

• Possibility of significant pain and distress
• Alternatives available
• Banned in most European countries
• Requires scientific justification for use in the
  U.S.

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Nontraditional Animals (relative replacement)

• Less sensitive to pain
• Lower procurement and maintenance cost
• Less public attention

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Limulus Amebocyte Lysate Test
Blood drawing from horseshoe crab
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Reduction

• Dissemination of relevant information
• Searching for the ideal animal model - transgenic
  animals, SCIDs
• Inbreeding
• Well-defined animal
• Experimental design- predictive computer
  comparison
• Statistics

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Refinement of Animal Well-being

• Environmental enrichment
• Improved methods of anesthesia and analgesia
• Non or less invasive procedures
• Humane methods of euthanasia
• Well-trained laboratory staff
• Minimize death-as-an-endpoint studies

Rabbit housing MSU ARC
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Advanced In Vivo Systems

• Transgenic animals

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Transgenic osteoporosis mouse
Osteoporosis
Normal
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Transgenics as Alternatives

• Advantages - viable substitutes for larger
  species Ex. Alzheimers, HIV, detection of
  mutagens and carcinogens
• Disadvantages - Large numbers are initially
  required and pain and distress may be associated
  with genetically induced diseases

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Definitions

• Comfort
• A state of physiologic and behavioral homeostasis
  in which an animal is adapted to its environment
• show normal feeding, drinking, and grooming
  patterns, social interactions, sleep/wake cycles,
  and reproductive activity.

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Definitions (cont.)

• Stress
• The effect produced by external events or
  internal factors that are referred to as
  stressors and that induce alteration in an
  animals adaptive state (homeostasis)
• Distress
• An inferred aversive state based on a variety of
  behavioral, physiologic, and psychological
  indices of an animals ability to adapt to the
  effect of stressors and the attendant stress.

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Definitions (cont.)

• Pain
• The sensation (perception) resulting from nerve
  impulses reaching the cerebral cortex via
  specific neural pathways (nociceptive pathways).
• Nociceptive is derived for Latin words meaning
  hurtful stimulus
• Nociceptors respond to excessive mechanical,
  thermal, or chemical stimuli

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Legal obligations

• AWA/PHS
• must avoid or minimize discomfort, distress, and
  pain
• PI must consider alternatives to procedures that
  might cause more than momentary or slight pain or
  distress
• Appropriate sedation, analgesia, or anesthesia
  must be used for procedures that cause more than
  momentary or slight pain or distress
• Potentially painful procedures must be planned in
  consultation with the attending veterinarian
• Neuromuscular blocking agents (paralytics) must
  not be used without anesthesia when performing
  painful or distressful procedures

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Pain Classification

• Superficial, deep, visceral
• Referred
• Psychogenic
• Acute, chronic

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Assessment of Pain

• Evaluate prior to experiment
• Will the animal feel pain (distress)?
• If yes, to what degree?
• What can be done to decrease or eliminate that?

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Recognition of pain

• Anthropomorphic criteria
• Species specific behaviors
• Abnormal behaviors that demonstrate anxiety and
  fear
• Pain scoring

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Recognition of Pain

• Exploratory behavior
• Food and water consumption
• Spontaneous locomotor activity
• Facial expressions
• Gait
• Reluctance to handling or palpation
• Vocalizations
• Mental status compare to previous behavior
• Change in activity levels
• Posture

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Methods of Pain Control

• Pharmacological
• Opiates (Narcotics)
• NSAIDS
• Environmental alteration
• Behavioral manipulation

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Euthanasia

• Greek terms eu meaning good and thanatos meaning
  death
• good death minimal pain and distress
• The act of inducing humane death in an animal
  (AVMA)
• Criteria are published in the 2000 Report of the
  AVMA Panel on Euthanasia

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Euthanasia

• Why is is necessary to euthanize animals that
  have been used for research?
• Many experiments require tissue examinations
  after the animals death
• Not appropriate to allow animals to be recycled
  through different experimental procedures

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Legal considerations

• AWA
• Must produce rapid unconsciousness and subsequent
  death without evidence of pain or distress
• Animals that would otherwise experience severe of
  chronic pain or distress that cannot be relieved
  will be painlessly killed at the end, or if
  appropriate, during the procedure
• PHS
• Method must be consistent with recommendations of
  the AVMA Panel on Euthanasia

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Humane considerations

• Quick and efficient, nonpublic area, away from
  other animals
• Describe criteria (endpoints) in advance
• Moribund animals
• Solid tumors
• Tranquilizers, sedatives, anesthetics, analgesics
• Professionalism and sensitivity of personnel
• Confirmation of death

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Human considerations

• Emotional distress experienced by the animal
  technician, as a result of euthanizing an animal,
  is perfectly normal response.
• Must cope effectively with euthanasia-associated
  emotional stress

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Methods

• Chemical
• Inhaled
• Injectable
• Physical
• Mechanical devices

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Methods

• The experience and skill of the animal technician
  is as important as the choice of method.
• IV injection
• Cervical dislocation
• Scientific justification
• Proficient in the technique

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Methods

• Euthanasia should not be done in the presence of
  other animals
• The procedure is not complete until the animals
  death has been carefully verified
• Heart stopped beating
• Respiration ceased

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Inhalant Chemicals

• Anesthetics
• Isoflurane, Halothane
• Mode of action CNS depression
• CO2
• Mode of action hypoxia of the brain
• Adv Rapid, Safe, Effective, no tissue residues
  or alteration of corticosteroids
• Disadv slow for neonates, question of
  pain/distress level

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Inhalant Chambers
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Injectable Chemicals

• General
• Vary widely in chemical composition
• Death can be induced by multiple routes
• IV preferred, most rapid and reliable
• IP often used in very small animals (rodents)
• Intracardiac must be anesthetized
• Consider pretreatment with analgesic,
  tranquilizer, or depressant

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Barbiturates

• Pentobarbital most common
• Preferred method for dogs, cats, large animals
• Mode of action CNS depression
• Adv rapid with minimal discomfort
• Disadv controlled, abuse potential

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Tricaine Methane Sulfonate (MS 222)

• Useful for anesthesia and euthanasia of
  amphibians and fish
• Buffer w/ sodium bicarbonate
• Concentration should be gt 250 mg/L
• Cannot be used for food animals

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Potassium Chloride

• Acceptable only in anesthetized animal
• Cardiotoxic
• Reduces risk of consumption toxicosis
• Not controlled substance

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Unacceptable Agents

• The following agents are unacceptable when used
  as the sole euthanatizing agent
• Strychnine
• Nicotine
• Caffeine
• magnesium sulfate
• neuromuscular blocking agents (succinylcholine,
  etc.)

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Physical Methods

• Humaneness dependent upon personnel skill
• Aesthetically displeasing
• Requires trained personnel and monitoring
• Personnel risks

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Physical Methods

• Cervical dislocation
• Poultry
• Mice or rats weighing lt 200 g
• Decapitation
• Guillotine
• Only used on conscious animals if it is essential
  for the research and IACUC has approved it

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Physical Methods

• Cervical dislocation
• Decapitation
• Microwave irradiation
• Penetrating captive bolt
• Stunning
• Gunshot

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Confirmation of Death

• Check respiration
• Exsanguinate
• Create pneumothorax