ILAR 40 (3)

Introduction: Animal Models of Pain. ILAR 40 (3): 095.
[Reviewer's Note - Any parts of this introductory article which recapped specific articles published in this ILAR issue were not reviewed as other LABSG members will be summarizing these articles for the LABSG list.] Acute pain is a commonplace experience (i.e. paper cut). Most animal experiments employ stimuli that produce acute pain of short duration and moderate intensity. These types of animal pain models have become standards in the screening of putative analgesics. Because animals are presented with short-duration, limited-intensity stimuli from which they can escape (i.e. withdrawal reflexes), these models are not "ethically" problematic. Most human pain conditions are not associated with limited-duration experimental stimuli but with waxing and waning persistent pain that is difficult to control. The American Pain Society estimates that 50 million Americans are partially or totally disabled by pain. Headache, head pain, and low back pain are common complaints. Pain is also associated with many human cancers. Although we know much about the anatomy, physiology, and neurochemistry of pain, not all pains are the same and consequently, not all pains are controlled well with the current treatment strategies. Investigators are developing animal models of persistent pain conditions seen in humans. Tissue injury and inflammation are commonly associated with various clinical conditions leading to persistent pain. Ethically, these models present challenges as current guidelines for the use of research animals try to avoid such scenarios. Many models of persistent pain employ inflammogens, which produce discomfort and are associated with hyperalgesia (enhanced response to a noxious stimulus). Visceral pain is distinct and different from pain arising from skin, muscles, and joints. Examples of persistent visceral pain include functional bowel disorders (i.e. irritable bowel syndrome, nonulcer dyspepsia) and interstitial cystitis. Postsurgical pain is also common but not always well controlled. Pain is no longer considered by the health care community as only a symptom, and long-lasting, chronic pain is in itself considered a disease or syndrome.
1. Which of the following statements concerning analgesia in rodents is TRUE?
A. Oral acetaminophen in a gelatin formulation can be used orally in rats and provides potent analgesia.
B. Buprenorphine in gelatin actually enhanced the inflammatory response and treated rats showed enhanced footpad swelling and lameness.
C. All of the above are TRUE
2. Which of the following HAS NOT been used as bolus parenteral analgesic therapy in rodents?
A. Flunixin meglumine
B. Phenylbutazone
C. Tricyclic antidepressants
D. Morphine
E. All of the above have been used
3. Which of the following is a potential disadvantage of using agonist-antagonist opioids for analgesia?
A. The "ceiling effect"
B. The "basement effect"
C. The "maximum effect"
D. The "minimum effect"
4. Which of the following would you expect to offer the least pain relief postoperatively?
A. Butorphanol
B. Oxymorphone
C. Buprenorphine
E. Morphine
5. Which alpha-2 adrenergics provide analgesia?
A. Xylazine
B. Detomidine
C. Medetomidine
D. Atipamezole
E. All of the above
F. A and C
1. B. 2. E. 3. A. 4. D. 5. F.

 Inflammatory models of pain and hyperalgesia. ILAR 40 (3): 111.
The article reviews the various inflammatory models of pain including persistent pain, mechanical sensitivity, orfacial inflammation, hyperalgesia vs. allodynia, and joint and muscle inflammation. It begins by comparing the injection of complete Freund's aduvant (CFA) and carrageenan into the hindpaw of rats to study the withdraw latency and duration (how long the paw is lifted) and paw licking and limb guarding responses to noxious stimuli. A shorter latency corresponded to a lowering of pain threshold. There is a rapid reduction of paw withdrawal latency which peaks in 2 to 6 hours persisting 1 to 2 weeks in CFA-treated rats.
Mechanical sensitivity is measured by measuring the total time the rats withdraw and hold their paw away from the test surface after two or more von Frey filaments are applied to the dorsal or ventral surface of the paw (measure of nocifensive behavior). Inflammation of the paw reduced the threshold and response duration (time from the start of the response to the return of the paw to the original position). Nocifensive behavior measurement of the orofacial region is performed through exposure to a radiant heat source and studying the head withdrawal. This method requires moderate training/acclimation to tesing environment/specially designed box. A low dose of pentobarbital helps to calm the animals during acclimation. All of these models provide the animal with control of the intesity or duration of stimulus. Other models (writhing response from injecting chemicals to study visceral pain) do not provide control to the animal or investigator.
Inflammatory Models of Persistent Pain discussion included cutaneous and subcutaneous tissues, joint inflammation and inflammation of muscle. Formalin injection into the footpad produces two phases of nocifensive behavior the first lasting about 5 minutes and the second more prolonged and persistant lasting about 40 minutes which includes shaking and licking of the paw. Activation of afferent fibers (first phase) and ongoing afferent activity produce the responses seen. Injection carrageenan, zymosan or CFA produces more persistent pain and hyperalgesia mimicking post-operative pain more closely. CFA produces the longest persistent pain (1-2 weeks). There are minimal decreases in grooming behavior and weight associated with these models.
Allodynia (result from sensitization fo peripheral nociceptive afferent fibers or mediated by low-threshold mechanoreceptive afferent fibers) and hyperalgesia (result from central sensitization and/or sensitization of nociceptive afferent fibers) are discussed as to their correct use when describing pain.
Intradermal capsaicin (model of neurogenic inflammation and hyperalgesia) evokes nocifensive behavior characterezed by lifting and guarding of the injected paw for 3 minutes and reduced withdrawal latencies lasting up to 45 minutes and mechanical stimuli persisting up to 4 hours. Other agents (mustard oil and zymosan are compared. Orofacial inflammation is studied by injecition formalin into the upper lip of the rat, mustard oil in the temporomandibular joint, or CFA into either area (which causes the most persistent pain). Fos expression increases in many nociceptive neurons in the dorsal horn after inflammation.
Joint inflammation (acute arthritis) induced by injection fo carrageenan and kaolin in knee joint under patella. CFA injection in the rat's tail can act as a model of polyarthritis which is a delayed hypersenitivity reaction occuring 10 days to 3 weeks later. This is, however, a systemic response causing multiple tissue lesions. Injection of urate crystals into the ankle joint can produce artritis within 24 hours with no systemic disease. Muscle inflammation can be induced by injection of carrageenan into the gastrocnemius, mustard oil or CFA in the deep massester muscle or tonque or hypertonic saline injection into the jaw to mimic jaw pain. Persistent injection of free-radical donor into the artery of the hindlimb or rats also produces an inflammatory response. Two new models include bone lesions in rats drilling a hole throught the tibia and systemic administration of an immunotherapeutic antiganglioside antibody producing a mechanical allodynia.
1) Which of the following inflammatory inducing agents produces the most persistent pain model?
A) formalin
B) mustard oil
D) hypertonic saline
E) carrageenan
2) Which of the following is used to quantify mechanical sensitivity?
A) head withdrawal latency using a specially designed box
B) von Frey filaments
C) writhing response
D) intradermal capsaicin
3) What method can be injected into joint to produce acute arthritis in 24 hours with no sytemic disease
B) capsaicin
C) urate crystals
D) mustard oil
C) free-radical donor
1) C
2) B
3) C

 Models of visceral nociception. ILAR 40 (3): 119.
Visceral pain is any pain arising from the internal organs,so examples are angina, colic, dyspepsia, and dysmenorrhea. There are some basic differences between pain in the surface of the body and visceral pain. In pain emanating from the surface of the body, it is well -localized, often evokes localized motor responses such as touching a hot stove and then withdrawing the hand. Visceral pain is not so well-localized, can cause a strong emotional response, can cause immobility coupled with tonic increases in muscle tone as well as non-specific autonomic changes in respiration, heart rate and blood pressure. The stimuli for the two types of pain is also different. Stimuli that routinely cause tissue damage( cutting, burning, and pinching) when applied to the skin always cause pain in the skin, but do not always evoke a painful response when the stimulus is applied to a visceral organ. Stimuli that frequently causes pain in visceral organs are often a higher intensity of a normal stimuli that causes no pain such as normal distention of the gallbladder versus over distention of the gall bladder.
There are three features required of a valid model of visceral pain.
The model must have a " noxious" visceral stimulus, the responses to that stimulus need to be reliable and reproducible. and there must be a different response to the noxious vs non noxious stimulus.
Visceral pain can be categorized into 4 groups, electrical stimuli, mechanical stimuli,
chemical stimuli, and ischemia.
In electrical stimuli, electrodes are placed on nerves which innervate visceral organs and pain has been evoked in humans. However, it is not very specific and therefore doesn't suit as a good model for visceral pain.
Mechanical stimuli can include probing or stretching but most commonly distends hollow organs with fluid or balloons. Mechanical stimuli fit the requirements for a good model of visceral pain It can be specific, quantified, isolated and controlled.
Chemical stimuli have been applied topically, intravascularly, or through physiological pathways such as cyclophosphamide induced cystitis. These can be good models depending on the particle chemical stimuli chosen.
Ischemia is occlusion of the vasculature, which then also produces a mechanical stimulus.
According to the classic definition by Sherrington in 1906, " a noxious stimulus produces or predicts tissue damage". In the visceral pain category, a noxious stimulus does not always cause tissue damage. Some people use the use the term
"algogenic" when referring to a noxious stimulus that ends in visceral pain.
This articles places the following results for a visceral stimulus to be termed noxious. These results are 1. causes pain in humans, 2. aversive behaviors in studied species 3. responses are modified with substances known to reduce visceral pain in humans
( morphine)
The term "nociception" is defined as " the perception of damaged tissue" and so the term nociception can be used in conjunction with nociceptive pain, because although there isn't always tissue damage, to the human or animal experiencing the pain it feels like there is. Reflex responses to noxious stimuli have been called pseudoaffective responses. These responses are profoundly affected by anesthesia suggesting that studies involving assessment of visceral pain in animals should be done on unanesthetized animals!
Many of the visceral stimuli to be described use surgical methods or are neither short in time period nor escapable. The model must prove to be valid before justifying the stimuli without anesthetics or analgesics. Neuro physiological responses to visceral stimuli have proved reliable, but due to the invasive surgery the animal must be anesthetized, spinally transected and/or decerebrated.
Due to the nonspecificity of most measured responses to visceral stimuli, a response to noxious visceral stimulus as a model of visceral nociception is considered valid if it is 1,. reliable (and reproducible) 2. not inhibited by compounds not considered analgesics 3 inhibited by analgesic manipulations know to reduce visceral pain in humans( morphine)
The paper goes on to review a number of models:
Writhing test: First described in the 1950's. A chemical irritant is injected IP and the subsequent number of " writhes" the animal makes is counted. Used primarily in rats and mice, described in primates, cats, dogs and guinea pigs. Phenyquinone or acetic acid are the most commonly used irritants. Other names for this test include, the abdominal stretch test, abdominal contortion test.
Test can be measured either as all or none for writhing or number of writhes counted per 5 minute for 30 to 60 minutes,. A third way to measure this test is to use a scale of 0-3. O being normal, and 3 being a classic " writhe". Some issues with the test, multiple nonanalgesics have inhibited writhing such as atropine and naloxone. There is also the ethical concern that is posed by a non anesthetized animal being given an inescapable noxious stimulus for 30-60 minutes. Used often, the writhing test does not meet the proposed criteria for a valid model of visceral pain.
Focal Application of Algesic Agents:
This is a modification of the writhing test by applying algesic substances to exposed surfaces such as epicardium or gall bladder.
A model from 1994 used a looped silicone catheter placed in the pericardial sac and then externalized 5 days before testing.
providing a mixture of bradykinin, acetylcholine, adensoine, histamine, serotonin and prostaglandin in awake rats. The rats exhibited passive avoidance behavior consistent with the stimulus being aversive.
Small Bowel Distention
1989- writhing responses that could be evoked in unanesthetized rats chronically implanted with a duodenal balloon.
The balloon distention without analgesics causes writhing, with morphine and other analgesics the rats exhibit normal
behavior. The actions of the rats indicate that the stimulus is aversive. In anesthetized rats distention of the duodenum, jejunum, and ileum evokes decreases in blood pressure. This model of visceral pain meets the criteria for a valid pain model, a similar stimulus causes pains in humans, it is aversive to rats, the reliable response is attenuated by known analgesics.
Colonic- Rectal Distention
Distention of the lower gi tract causes many reliable and reproducible responses in species including, horses, dogs, cats, rabbits and rats. In rats cardiovascular responses to phasic, constant -pressure colonic distention ( CRD) has been demonstrated to be reliable and reproducible. A balloon is used to cause the colonic distention. CRD also fulfills the stated criteria for a valid model of visceral pain.
Biliary Distention. In cats, ferrets and primates the common bile ducts have been cannulated or ligated to cause distention.
Cats and dogs have shown "escape' behavior with a distended gall bladder. At the time the article was written (1999) the reliability and reproducibility of this stimulus had not been determined.
Artificial Kidney Stones
After surgical exposure, an artificial stone is placed in the upper third of the ureter using dental cement. Rats are recovered and
observed for 4 to 14 days. Rats are monitored for " visceral episodes" similar to writhing. This is a fairly reliable model of visceral pain, but can vary between animals so significant skill is required in conducting these experiments. This model also raises ethical concerns because the noxious visceral stimulus is inescapable and of long duration.
Urinary Tract Distention
In 1982 Brasch and Zeller characterized hemodynmaic responses to renal pelvis distention in the pentobarbital anesthetized rat. Only a few studies have utilized the bladder distention model of pain. Instead more studies work at evoking the micturation reflexes.
Urinary Bladder Irritants
Experimentally, animals have had turpentine, mustard oil, croton oil, acetic acid, acetone, xylenes, capsaicin placed in the bladder to cause irritation. Most studies have been in rats, although primates and cats have been used. McMahon and Abel reported the use of a chronic decerebrate rat model, minimizing the ethical concerns about the painful stimulus. There are two other models for bladder irritants. A laparotomy is performed 24 hours before the test and a 1 mm diameter cannula is placed in the bladder. The next day the rats receive an injection of xylem through the catheter. Immediate behavioral responses such as head turning, licking of abdomen, biting, vocalizing, etc. are seen. A third model is used to mimic the clinically noted pain due to cystitis from anti-neoplastic agents. In this model rats are dosed with cyclophosphamide IP, and rats are monitored for aversive behavior.
Reproductive Organs
Studies have been done with distention or pressure on uterus, vagina and cervix of rats.
Limited studies have been done with testicular compression. Not enough has been done in this area to evaluate the models for visceral pain models.
Ischemic Stimuli ( Coronary Artery Occlusion)
Cessation of blood supply to most viscera leads to pathologic pain and morbidity and mortality. Coronary artery insufficiency
in humans leads to angina( heart pain). However ischemia produces responses that are not reliable." Silent ischemia" is defined as a significant episode of coronary artery occlusion and ischemia with no pain. Cardiovascular responses to occlusion can be highly variable even in the same study. The variability of responses to ischemic visceral stimuli has been proposed to be related to the instinsic neural circuitry within visceral structures and to interactions of vagal, brainstem and sensory components.
At this time ischemic stimuli is not a good model for visceral pain.
Model of visceral inflammation: Miampamba(1994,6) injected formalin into the colonic wall of briefly anesthetized rats a
and then observed the rats over three hours for 4 behaviors.
Model of pancreatitis: Houghton( 1997) validity has yet to be determined, but may have significant clinical relevance.
No questions

 Postoperative models of nociception. ILAR 40 (3): 129.
Postoperative pain is a common form of acute pain, and it is commonly thought that it can be adequately treated with parenteral opioids. However, studies of patients after major surgeries indicate that postsurgical pain induced by activity is poorly responsive to opioids compared to pain at rest. Pain research is rarely conducted in this area. Mechanical hyperalgesia (a decreased pain threshold and an increase in pain response to suprathreshold stimuli) is a properly of incisional pain. Previously there has been no animal model for incisional pain. This paper describes a novel rat model to assess incisional pain.
Rats were anesthetized and a longitudinal incision is made on the plantar surface of the hind foot. The plantaris muscle is elevated and incised longitudinally. The wound is closed with mattress sutures. Pain-related behaviors were assessed using calibrated Semmes Weinstein von Frey filaments. Each filament is applied once starting with 15 milliNewtons and continuing in an ascending order until a withdrawal response occurs or 522 mN (the cutoff value) is reached. Using this system, it was determined that mechanical hyperalgesia persists for several days after the incision and then gradually decreases. Other testing modalities included applying a blunt mechanical stimulus (plastic disk attached to a nylon monofilament) to the incision. The ability to ear full-weight on the surgerized foot was also evaluated.
The significance of this model lies in that it better assesses true post-operative painfor several reasons.
1. Surgical incisions produce tissue injuries that are distinctly different from inflammatory pain, chemical irritation or nerve injury.
2. Testing modalities for pain-related behaviors used in this study are a better measure than traditional nociceptive measures (tail flick test). Thermal hyperalgesia may or may not be related to mechanical hyperalgesia.
3. The time scale of when incisional pain occurs is similar to most postoperative human patients. Traditional models have onsets varying from minutes (formalin) to days (nerve injury).
Other postoperative pain models have included ovariohysterectomy in rats. This model has obvious relevance to a common clinical procedure and the injury includes peritoneal and visceral injuries. The same procedure has been studied in dogs. A model for investigating bone pain has been described. A hole is drilled through the tibia or calcaneus in the rat.
1. What is a von Frey filament?
2. What is mechanical hyperalgesia?
1. A von Frey filament is a nylon monofilament with varying diameters. As the diameter of the filament increases, the force necessary to bend the filament increases. These monofilaments are applied individually until a withdrawal response occurs.
2. Mechanical hyperalgesia is a decreased pain threshold and an increase in pain response to suprathreshold stimuli.

 Ethics and Pain Research in Animals. ILAR 40 (3): 097.
Pain is an evil in and of itself. Pain can have additional bad effects and for those reasons too be considered evil, but pain is evil simply because it feels the way it does. From this fact several ethical principles follow:
1. The "Equality Principle" holds that a given amount or duration or severity of pain is equally an evil for any being-human or animal.
2. The "Justification Principle" states that because pain is an evil, anyone who causes pain in a being that can experience it must show that it is necessary and justify to cause this pain.
3. The "Value Principle" states that the more pain an experiment or test will cause, the greater must be it's value. The value of research causing pain must be greater when the pain for the animals is worse. There are 2 components of the kind of value that is needed to justify animal pain caused by research: 1. The value of the animal research and 2. The level of scientific soundness. Bad science cannot be good ethics.
4. The "Minimization Principle" holds that we should minimize pain experiencd by research animals. This is in reality a difficult principle to apply because there are many components ie: duration, severity, and characteristics ie: dull, piercing. Is one minute of sharp pain worse than 10 minutes of dull pain? The underlying motivation is that animals feel no worse than necessary.
While some believe that it is acceptable to subject a few animals to more pain than is justifiable so that other animals will feel no pain, the author suggests that fairness to individual animals may require using more rather than fewer animals so that each animal experiences the minimal amount of pain possible. The frequent invocation of the Minimization Principle in AWA regulations and PHS policies reflects the centrality of this principle in society's ethical framework relating to animals.
While the ethical principles relating to pain discussed above are widely accepted and seem reasonable, there is imprecision in applying them because animals cannot talk and there is still much to learn about what behavioral and physiological signs indicate the presence of pain in various species. It is therefore more accurate to say that one's ethical obligation regarding animal pain is not to minimize pain but to try and minimize it to the best of our knowledge.
The following are Ethical Guidelines for IACUC's:
1. The IACUC should apply to the consideration of any pain research proposal the fullest and most complete consideration available. Pain research proposols should not be expedited or reviewed by delegated member, committee, or subgroup of the committee. It is especially important that non-affiliated members be present to review pain research protocols.
2. Any proposol of pain research should have clear and convincing statements of the justification and value of the research. The greater the pain, the greater must be the justificaiton and value of the research.
3. Similar to #2 just stated a different way.
4. Investigators should characterize and estimate the likely pain and associated negative feelings that will be experienced by the animals as completely and accurately as reasonably possible. Investigators and IACUCs should consider a wide range of evidence, including inferences from similar pain experiences in humans and the best available scientific data regarding behavioral and physiological signs of animal pain.
5. Investigators should assure the IACUC that they are attempting to minimize pain in the design of the resarch. Investigators should bear the burden of demonstrating to the IACUC why pain-relieving tools(analgesia, pain avoidance, environmental enrichment), cannot be used consistent with experimental aims.
6. In balancing pain and distress caused to animals against the value of the research, and in monitoring pain research in progress, the IACUC should consider whether pain has become so severe that individual animals should be removed from the research or the research itself should be terminated.
7. Pain in "lower" species may not be considered less harmful or in need of less justification than pain in "higher" species.
8. Investigators and IACUCs should focus on what is fair to individual animals, which may sometimes require lengthening rather than shortening the duration of pain, using more rather than than fewer animals, or using more rather than less total pain.
9. IACUCs should attempt to adhere to government ethical rules and should consult relevant professional association ethical guidelines but should view all general rules and guidelines as calling for, and ultimately subject to, independent ethical deliberation.
The author continues to outlines the Ethical Guidelines of various professional groups. Many of these documents explicitly incorporate or refer to US government laws and regulations.
1. What is the federal law that insures that animals used in research facilities or used for exhibition are treated humanely?
2. The author states two necessary components of the kind of value that is needed to justify animal pain caused by research.
3. What are the four ethical principles that stem from the fact that pain is an evil in and of itself. Which of these principles is the most difficult to employ.
4. How does the author feel about inflicting more pain than is necessary on a few animals to spare other animals from experiencing any pain?
1. The Animal Welfare Act
2. 1. Value. 2. Scientific Merit.
3. 1. The Equality principle
2. The Justification Principle
3. The Value Principle
4. The Minimization Principle
The most difficult principle to employ is the Minimization Principle, because of the imprecision of pain determination in animals.
4. The author feels it is not fair to the individual animal that is experiencing more pain. The author feels having more animals on a pain research project is better if by doing so each animal experiences less pain.