Pain in Fish

1
Pain in Fish?

• Jennie Lofgren DVM, MS
• MIT Division of Comparative Medicine
• 2/1/08

2
Overview

• Why does it matter?
• Approaches to discussion
• Neuroanatomical
• Physiological
• Behavior
• Current guidelines and recommendations.
• Conclusion
• References

3
Why does it matter?

• Roughly 2.8 million fish are used for research
  and education in the United States each year.
• Fish represent nearly 25 of all animals used for
  research and education in North America only
  mice are used in higher numbers.

Grey and Vincent, 2006.
4
Why does it matter?

• The number of fish being used in research is
  increasing due to
• Ethical awareness phylogenetically lower
  species
• replacement for warm-blooded animals.
• Compared to transgenic mice, inexpensive and easy
  to maintain.
• Ideal species in which to study carcinogenesis,
  renal and cardiac regeneration, environmental
  toxicology and proteomics.

Borski and Hodson, 2003.
5
Why does it matter?

• Zebrafish have a well characterized genome.
• According to the Foundation for Biomedical
  Research, Zebrafish are one of the most promising
  models for studying early vertebrate development
  and gene function.

Grey and Vincent, 2006.
6
Why does it matter?

• Common techniques used in research with fish
  include fin, gill, or organ biopsies, skin
  scrapings, and tissue or cell implantation.
• With the increasing use of fish, IACUCs have
  begun to struggle with the question of pain and
  distress in fish and what they should require of
  investigators.

7
Approach to Discussion

• The academic debate over whether fish experience
  pain is well documented.
• Recent literature can be divided into three main
  approaches
• Neuroanatomical
• Physiological
• Behavioral

8
Neuroanatomical

• Fish share some but not all of the
  neuroanatomical structures utilized in human pain
  perception.
• Does this then mean that they cannot feel pain?

9
Neuroanatomical

• 2 main aspects of pain perception
• Nociception conversion of a noxious stimulus
  into an electrical impulse appreciated at the
  spinal level and forwarded to the brain.
• Conscious and emotional appreciation of pain
  providing a suffering component.

10
Nociception

• C fiber neurons unmyelinated
• long-lasting "noxious pain"
• associated with longer-term, dull, aching,
  throbbing pain.
• A delta fiber neurons myelinated
• immediate "adaptive pain," such as that
  experienced milliseconds after slamming your
  fingers in a door.
• Both of these fiber types were identified in the
  trigeminal nerve of trout.

Sneddon, Braithwaite, and Gentle. 2003a, 2003b.
11
Neuroanatomical
Primary sensory cortex
Limbic system, which includes the amygdala.
Thalamus
Second-order nocioceptive neuron that crosses the
midline and ascends up the spinothalamic tract
http//www.mayoclinicproceedings.com/inside.asp?AI
D795UID
Nocioceptive fibers (A and C) cell bodies are
in the Dorsal root ganglion (DRG)
12
Neuroanatomy a tale of 2 theories
13
Neuroanatomy a tale of 2 theories

• Theory One
• Consciousness is a prerequisite for pain
  suffering.
• Consciousness requires a neocortex.
• Fish lack a neocortex, therefore they are
  incapable of consciousness and cannot appreciate
  pain nor suffer.

Neocortex
Rose, 2002, 2007.
14
Neuroanatomy a tale of 2 theories
Forebrain (cerebrum, telencephalon)
Hind brain (brainstem)
Mid brain (diencephalon)
Rose, 2002.
15
Neuroanatomy theory 1

• Nightmare (human)
• elevated heart and respiratory rates
• increased blood pressure
• release of stress hormones (cortisol)
• dilated pupils
• may even scream or thrash
• Not conscious- cannot be in pain nor suffer.
• How would an IRB approach a study that, as a
  sequelae, induced nightmares?
• Would an intervention be preferential if it would
  prevent the nightmare ?

Rose. 2002.
16
Neuroanatomy a tale of 2 theories

• Theory 2 3 parts.
• First Homologous structures with mammalian
  neuroanatomy.
• Fish telencephalon with mammalian limbic system.
• Limbic system anticipation, expectation, and
  ultimately goal directed activity.
• Amygdala (component of the limbic system)
  emotional response and memory.
• Only brain component consistently demonstrated to
  respond during conscious experience of pain in
  humans as demonstrated with imaging studies.

Chandroo, 2004a, 2004b.
17
Neuroanatomy a tale of 2 theories
18
Neuroanatomy a tale of 2 theories

• Theory 2
• Second
• Dynamic core hypothesis
• Consciousness is not the function of a single
  structure with strict neuroanatomical boundaries
• Consciousness is generated through shifting
  functional connectivity of neurons.

Chandroo, 2004b.
19
Neuroanatomy a tale of 2 theories

• Theory 2
• Third
• Neuroanatomical development in fish is extremely
  different from that of humans
• The ability to definitively identify structures
  responsible for consciousness in fish have yet to
  be determined.

Rodríguez, Broglio, Durán, Gómez, and Salas.
2006. Broom. 2007.
20
Neuroanatomy a tale of 2 theories

• Third
• Ex
• mammalian neural tube folds in on itself
• the bony fish neural tube folds out
• in comparison to a mammalian brain, the neural
  structures of the fish forebrain are reversed in
  order
• Ex
• fish brains put greater emphasis on different
  stimulus that mammals (lateral line vs. sight)
• change in priorities is reflected in the
  composition of the brain.

Rodríguez, Broglio, Durán, Gómez, and Salas.
2006. Broom. 2007.
21
Neuroanatomy

• One definitive statement made by The Fisheries
  Society of the British Isles.
• The lack of a neocortex does not mean that fish
  cannot experience some kind of suffering. Recent
  studies suggest that fish have the capacity to
  perceive painful stimuli and that these are
  strongly aversive states that we associate with
  pain and emotional distress.

Fisheries Society of the British Isles. 2002.
22
Physiology

• Stress
• Neuroendocrine
• Response to exogenous drugs
• Anxiolytics
• Analgesics

23
Stress

• Acute stress can an adaptive mechanism which aids
  in survival
• Chronic, unavoidable stress is detrimental
• leads to increased illness and generally poor
  welfare

Huntingford. 2006.
24
Stress

• Hypothalamic-pituitary-interrenal response
• almost identical to the mammalian
  hypothalamic-adrenal-pituitary axis.
• Elevated cortisol is an indicator of fish
  welfare.
• Measured
• invasively through a blood stick
• non-invasively in tank water or feces.

Iwama. 2007. Chandroo. 2004a. Huntingford. 2006.
25
Stress

• Study
• Salmon conditioned to tank emptying
• demonstrated by decreasing amounts of secreted
  cortisol
• On transportation, the conditioned fish had a
  higher survival rate than those that had not been
  conditioned.

Chandroo. 2004a.
26
Stress

• Chronic stress has been demonstrated in
  subordinate fish.
• More closely related to threat imposed by the
  presence of the dominant fish rather than actual
  aggressive encounters.
• Appreciation of danger without having experienced
  actual physical insult.
• Demonstrates fear?

http//en.wikipedia.org/wiki/ImageGeorgia_Aquariu
m_-_Giant_Grouper_edit.jpg
Chandroo. 2004a.
27
Neuroendocrine

• Substance P
• peptide found in mammals
• known to modulate and transmit pain
• identified via immunohistochemistry in the fish
  brain.
• FMRFamide
• peptide neurotransmitter found in mammals
• functions in analgesic and aversive responses
• identified in the trout brain.

Chandroo. 2004a.
28
Neuroendocrine

• Serotonin
• In human brains is naturally produced in the
  limbic system (mediates psychological and social
  stress)
• In fish, serotoninergic activation in the
  telencephalon (homologous to the mammalian
  limbic system) identified during socially
  stressful events - being exposed to a dominant
  fish.

Chandroo. 2004a.
29
Dopamine

• Mammals influence learning and behavior
  resulting from emotion
• Fish exposure to dopamine agonists
• amphetamine (intracranial injection)
• apomorphine (immersion)
• demonstrated dose dependent changes in reward
  behavior and reduced aggression

Chandroo. 2004a.
30
Benzodiazepam

• Fish given anti-anxiety medications that bind to
  benzodiazepine receptors in the brain secreted
  less alarm pheromone.

Chandroo. 2004a.
31
Opioids

• Opioid receptors have been located in the
  zebrafish limbic structure.
• Multiple studies have demonstrated intriguing
  affects of opioid administration.

Chandroo. 2004a.
32
Opioids Study 1

• Goldfish withstood increasing electric shocks as
  morphine was added to their tank water.
• Effect was reversed when opiate antagonists were
  administered.
• Criticism- possible that the goldfish were not
  alleviated of pain but rather their mental
  capacity was diminished in the presence of
  opioids.

Ehrensing, Michell, Kastin . 1982.
33
Opioids Study 2

• Carp given butorphanol or a sham dose IM
  post-operatively
• Sham dose
• displayed decreased activity
• lower in the water column
• decreased feeding intensity
• Butorphanol
• no significant alterations from pre-surgical
  behavior i.e. retained normal behavior.

Sneddon. 2003a.
34
Opioids Study 3

• Normal fish behavior neophobic
• Fish avoid a new object placed in the center of
  tank
• Fish administered acetic acid (an irritant)
• Spent more time closer to the novel item
• Avoidance behavior was greatly reduced
• Given morphine
• Regained normal avoidance behavior

Sneddon. 2003a.
35
Behavior

• Asks fish to demonstrate desire to avoid pain
  and distress through preference testing.
• Insight into an animals priorities.
• Measure of motivational affected state.
• Assumes that given a choice between discomfort
  and comfort, an animal will not freely choose
  discomfort.

Volpato. 2007 Dunlop, Millsopp, Laming.
2006. Braithwaite, Boulcott. 2007.
36
Behavior

• Are the fish are making a choice or just
  responding reflexively?
• Two studies in particular address this concern.

37
Reflex vs. Choice Study 1

• Simulated predator presented when fish swam
  across the median of the tank
• Elicited a fast start response
• Fast start behavior is a documented reflex in
  fish seen in feeding behavior and in response to
  predators.

Chandroo. 2004a.
38
Reflex vs. Choice Study 1

• Fish soon changed their behavior
• avoided the center of tank.
• if forced to swim through the center of the tank
  fish adopted a zig-zag motion (evasive but not
  reflexive maneuver).
• Study conclusion fish anticipated a
  frightening stimulus and developed an escape
  response that was motivated by an affective state
  of fear.

Chandroo. 2004a.
39
Reflex vs. Choice Study 1

• If fish were only capable of a reflexive response
  they would display repetitive behavior fast
  start response over and over without the
  possibility of behavior modification.

Chandroo. 2004a.
40
Reflex vs. Choice Study 2

• Trout placed in a shuttle tank.
• Exposed to plunging net.
• elicited a fast start response (reflex) to
  shuttle to the safe chamber.
• A light was then shown prior to the net.
• After conditioning
• When shown the light, fish would shuttle at a
  slower (non-reflex) pace to the safe
  compartment.

Yue. 2004
41
Reflex vs Choice Study 2

• Conclusions
• trout recognized the light as a neutral stimulus.
• presence of the light initiated behavior that
  would allow avoidance of the negative stimulus,
  the net
• Author extrapolated that trout were less stressed
  when allowed to adopt an effective strategy to
  avoid negative stimuli.

Yue. 2004
42
Behavior

• Study how intensely fish prefer or dislike a
  given experience through
• complex cognitive abilities
• stress induced changes in memory

Volpato. 2007.
43
Cognitive Ability

• Trout and goldfish trained to associate areas of
  a tank with increasing levels of electric shock.
• Both species learned to avoid areas of the tank
  associated with shock.
• In 2nd part of the study, fish were shown a
  conspecific in the area of the tank with greatest
  shock.

Dunlop, Millsopp, Laming. 2006
44
Cognitive Ability

• Trout chose to sustain mild electric shock to be
  closer to their companion.
• Goldfish chose to stay as close to the
  conspecific as possible without sustaining any
  shocks.
• Neither chose to be in the area of the tank
  furthest from both the shock and conspecific.
• May demonstrate that fish can choose what level
  of negative stimuli is acceptable given the
  possibility of positive stimuli.

Dunlop, Millsopp, Laming. 2006
45
Stress, Memory, and Behavior

• Declarative memories
• A memory with a specific significance attached to
  it (positive or negative), can be recalled to
  influence future behavior.
• Behavior gives insight into the internal state of
  the fish while it was experiencing a stimulus.

Chandroo. 2004 Yue. 2004.
46
Stress, Memory, and Behavior

• Paradise fish
• recognize a given conspecific for 7 days
• encounter with a fish of another species is
  remembered for 3 months
• Carp
• pass up bait for up to 3 years after being hooked
  just once.

Yue. 2004
47
Stress, Memory, and Behavior

• Siamese Fighting Fish
• Shown a fight between two fish
• If then put in the same tank as the winner they
  were less likely to engage in battle
• If the put in the same tank as the loser were
  more likely to engage in battle
• Rainbow trout
• Recognize past opponents
• Will change their fighting behavior depending on
  the outcome of the prior fight

Chandroo. 2004
48
Behavior Studies

• Provides an alternative to the notion that fish
  are merely responsive, passively reacting to
  stimuli as they encounter them with little or no
  ability for cognition.

Braithwaite, Boulcott. 2007.
49
Current Guidelines

• On going debate regarding pain in fish
• Current guidelines are allusive and require
  individual interpretation.
• Fish are not covered under the Animal Welfare
  Act.

Animal Welfare Act as Amended in United States
Code, Title 7, Sections 2131 to 2156. (1996).
50
PHS Policy

• covers all vertebrate species
• Requires
• avoidance or minimization of discomfort,
  distress, and pain when consistent with sound
  scientific practices
• Unless the contrary is established,
  investigators should consider that procedures
  that cause pain or distress in human beings may
  cause pain or distress in other animals.
• Procedures with animals that may cause more than
  momentary or slight pain or distress should be
  performed with appropriate sedation, analgesia,
  or anesthesia

Office of Laboratory Animal Welfare of the
National Institutes of Health. PHS Policy on
Humane Care and Use of Laboratory Animals. Public
Health Service. p 27. (1986).
51
American Fisheries Societies Guidelines for the
use of Fishes in Research (1988)

• Recommended
• application of a local anesthetic, such as
  benzocaine, to any area that will undergo tissue
  removal or modification
• general anesthesia during prolonged restraint

American Fisheries Society (AFS), American
Society of Ichthyologists and Herpetologists
(ASIH), and American Institute of Fishery
Research Biologists (AIFRE). AFS Policy Statement
16, also published as Guidelines for Use of
Fishes in Field Research. Fisheries. 13(2),
16-23 (1988).
52
American Fisheries Societies Guidelines for the
use of Fishes in Research (2002)

• Primarily cites the Rose 2002 paper as the basis
  for discussion of pain.
• Fish are capable of experiencing physiological
  stress and nociception but not pain or suffering
• Recommendations are focused on reducing
  physiological stress through good husbandry
  practices.

http//www.fisheries.org/afs/publicpolicy/guidelin
es2004.pdf (2004).
53
European Regulations

• Fisheries Society of the British Isles
• Pain in fish is not only possible, but likely.
• Recommendations encompass good husbandry
  practices and preventing or addressing
  potentially painful stimuli.
• According to an article discussing the German
  animal welfare law, fish are capable of feeling
  pain and they are able to suffer...

Fisheries Society of the British Isles. Fish
Welfare. Briefing 2. Granta Information Systems,
82A High Street, Swanston, Cambridge CB2 4H.
2002 Oidtmann, Hoffmann. 2001.
54
Veterinary Texts

• Admit that there is no clear answer
• Recommend using analgesia as there is evidence
  that it is possible that fish feel pain.

Brown. 1992 Machin. 2001
55
Recommendations from Veterinary Texts

• Anesthetics
• Benzocaine, lidocaine, and tricaine
  methanesulfonate (MS-222) in tank water
• also provides localized analgesia
• recovery from anesthesia requires removal from
  immersion
• the analgesic effect is only present while the
  fish is in the anesthetic solution

Machin. 2001 Carpenter. 2005.
56
Recommendations from Veterinary Texts

• Morphine, dissolved in tank water, has
  demonstrated analgesia in response to electric
  shock.
• Butorphanol has also been given intramuscularly
  (IM) for post operative analgesia.

Machin. 2001 Carpenter. 2005
57
A special note on opioids

• Fish are ectotherms.
• Study the same dose of morphine was increasingly
  efficacious with decreasing temperature of the
  fish.
• As the fishs temperature drops, the blood pH
  increased, which increased the amount of free
  base drug.
• Therefore, the temperature of the fish should be
  considered when deciding on an appropriate dose.

Stevens, Balahura. 2007.
58
Recommendations from Veterinary Texts

• Ketamine, alone or in combination with
  medetomidine, have been known to provide both
  anesthesia and analgesia in fish.
• The medetomidine can be reversed with atipamezole
  (Antisedan, Pfizer).

Carpenter. 2005
59
Studies using analgesia

• Koi carp
• given 0.4mg/kg butorphanol IM intra-operatively
• experienced significantly reduced recovery time
  compared to the control group (not given pain
  medication).

Harms. 2005.
60
Studies using analgesia

• Carp
• Ketoprofen at 2mg/kg IM intra-operatively
• significantly lower post-surgical creatine kinase
  levels
• did not have significantly different
  post-operative recovery time compared to the
  control group
• Ketoprofen at this dose did not provide
  sufficient analgesia.

Harms. 2005.
61
Studies using analgesia

• Carp
• Tramadol at 10 and 100 nmol/g IM
• achieved statistically significant analgesia
  compared to carp without pain medication
• Analgesia measurable within 5-15 minutes
• Analgesic effect was stable for 1-2 hours.
• Naloxone at 100nmol/g blocked the analgesic
  effect.

Chervona, Lapshin. 2000.
62
Studies using analgesia

• Non-opioid analgesics effective in amphibians.
• Chlorpromazine, chlorodiazepoxide (a
  benzodiazepine), and diphenhydramine (Benadryl)
  were roughly 50 as affective as morphine.
• NSAIDs such as indomethacin and ketorolac were
  roughly 40 as effective as morphine.
• May be useful in fish.

Stevens, Maciver, Newman. 2001.
63
Table 1. Analgesic Agents in Fish
Machin. 2001., Carpenter. 2005.
64
Summary

• Research using fish as animal models has
  demonstrated great promise.
• Their increasing use is bringing with it a need
  to understand their experience as research
  animals.

Hirschler. 2007. Snyder. 2006.
65
Summary

• Minimizing pain and distress is an inherent
  responsibility with animal use.
• Fulfilling this responsibility is especially
  difficult in a species so different from mammals.
• Studies designed to answer the question of
  whether fish feel pain and therefore require
  analgesia fall into three main categories
• the neuroanatomical capabilities of fish
• their physiological responses
• behavioral adaptations to stress and pain

66
Summary Neuroanatomy

• Fish are capable of nociception.
• Do not possess a neocortex which some believe is
  solely responsible and required for
  consciousness.
• Possible through homologous structures or several
  structures acting in concert, that fish
  experience some form of conscious pain.

67
Summary neuroanatomy

• No definitive studies to determine if fish
  possess the neuroanatomy necessary to consciously
  perceive pain.
• Advanced imaging and molecular techniques may
  allow visually identification of changes in the
  fish brain in response to noxious stimuli.

Huntingford, 2006. Braithwaite, Boulcott. 2007.
68
Summary Physiology

• Fish share much of the same physiological
  responses to stress and pain as mammals.
• Most interestingly, fish seem to benefit from
  post operative analgesia.

69
Summary Behavior

• Demonstrated difference between reflexive
  response and escape behavior from aversive
  stimuli.
• If fish have the choice to avoid danger or pain
  they do so.
• Ability to weigh positive and negative stimuli
  and adjust their behavior accordingly.

70
Conclusion

• It may not matter whether fish are able to
  consciously experience pain in the human sense.
• There are a myriad of studies that conclusively
  demonstrated that stimulation that would be
  painful in a mammal can be extremely aversive to
  fish.
• As well as evidence that fish seem to benefit
  from pain relief.

71
Conclusions

• It is the responsibility of scientists,
  veterinary and animal care staff committed to
  excellent stewardship and welfare of laboratory
  animals to minimize experiences that would cause
  pain and distress.

72
References

• Diseases of Aquatic Organisms
• Special 2 Welfare of Aquatic Organisms
• May 2007.
• Open Access articles
• http//www.int-res.com/abstracts/dao/v75/n2/

73
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• 35. Snyder, Alison. Secret of Heart Regeneration
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Questions?