Animal Welfare Information Center Newsletter, Winter 1997/1998, Vol. 8, no. 3-4 *************************

A Whole-Animal Alternative Model for Pain Research

by
Craig W. Stevens, Ph.D.
Department of Pharmacology and Physiology
Oklahoma State University-College of Osteopathic Medicine, Tulsa, Oklahoma


Alternative models for biomedical research seek to address refinement, reduction and/ or replacement of existing animal models for reasons of ethical considerations. Alternative models must also be founded in sound scientific rationale and able to compete for scarce research funds. This brief review describes a unique alternative model using live amphibians for research into opioid analgesia and more generally for pain research.

[PHOTO:  The Northern grass frog, Rana pipiens.] The ethical basis for an amphibian model stems from a comparative neurological approach to the study of pain and analgesia, which will be described briefly below. (For simplicity, the terms pain and analgesia are applied to nonhuman animals; some would prefer the more precise terms nociception and antinociception.) At present, we have an active research program using the Northern grass frog, Rana pipiens, to investigate the analgesic actions of opioid [6, 10, 15-18] and alpha-adrenergic drugs [2, 13]; morphine tolerance [14]; and stress-induced analgesia mediated by endogenous opioid peptides (endorphins) and the action of enkephalinase-inhibitors [19]. However, the focus of this review is on the ethical and scientific aspects underlying development of the amphibian model, rather than a discussion of our research results. For a more exhaustive review of nonmammalian models for pain research, see elsewhere [12].


To: [Introduction] | Why Do Pain Research | Special Nature of Animal Use in Pain Research | A Case of Comparative Substitution Using Amphibians | Studies of Opioid Analgesia Using Amphibians | Conclusions | Acknowledgments | References

Why Do Pain Research?

There is no question that use of mammalian models for pain and analgesia research has led to tremendous advances in the understanding of nociceptive transmission, the actions of analgesic drugs, and the function of endogenous opioid systems. Over the past two decades, advances from the biomedical research community have been translated into effective therapeutic interventions for millions of patients suffering from acute and chronic pain syndromes. However, there are a number of pain-related disorders that remain refractory to successful clinical treatment, such as neuropathic pain, and complications of current pharmacotherapy, such as opioid tolerance and dependence, warrant continued research into the basic mechanisms of pain and analgesia using animal models. Additionally, there is an ongoing need for use of animal models in efficacy and safety testing of new analgesics in the pharmaceutical industry.
To: [Introduction] | Why Do Pain Research | Special Nature of Animal Use in Pain Research | A Case of Comparative Substitution Using Amphibians | Studies of Opioid Analgesia Using Amphibians | Conclusions | Acknowledgments | References

Special Nature of Animal Use in Pain Research

Unlike most animals used in biomedical research, pain and analgesia researchers use whole behaving animals so that anesthetics or analgesics cannot be administered. As we have no access into the sensorium of an experimental animal, a behavioral test for measuring analgesia must be used. Most analgesic tests used in mammals are self-limiting such that the animal responds to the noxious stimulus and the stimulus is terminated (stimulus-control by the animal). For example, in the rodent tail-flick test, the behavioral response of a mouse or a rat is observed following the presentation of a thermal stimulus and analgesia is measured by the time it takes for the rodent to flick its tail off a projector lamp. The hot plate test entails placing a rat or mouse on a heated surface, usually about 55o C, then measuring the time it takes for the animal to jump or lick its hindpaws. In both cases, the latency to the endpoint is taken before drug or experimental treatment and again at various times after treatment. Other acute analgesic tests include the paw-pressure test and the paw withdrawal from a focused heat source. This last analgesic test is often used in studies of chronic pain in which one hindpaw will be injected with a pro-inflammatory substance (for example, formalin) and the contralateral side will be used as a control. Finally, these types of studies are also done using cats, dogs, and primates, although to a much lesser extent.

Over the past 40 years, thousands of analgesic drugs and treatments have been tested using mammalian models in this way. More recently, the development and popularity of a number of chronic pain models in mammals raises additional ethical issues as there is the possibility of persistent pain in mammals without the ability to terminate the noxious stimulus. The duration of potential pain and its escapability are important ethical considerations for researchers and are included in the guidelines for use of animals in pain research [23].


To: [Introduction] | Why Do Pain Research | Special Nature of Animal Use in Pain Research | A Case of Comparative Substitution Using Amphibians | Studies of Opioid Analgesia Using Amphibians | Conclusions | Acknowledgments | References

A Case for Comparative Substitution Using Amphibians

[PHOTO] [PHOTO] [PHOTO]
Figure 2. Routes of drug administration in frogs. Left, systemic administration into the dorsal lymph sac. Middle, spinal administration by direct percutaneous intraspinal injection (i.s.). Right, supraspinal administration b y injector placed in the third ventricle of the brain (i.c.v.). See text for further details.

Ethical considerations. Comparative substitution was defined by Russell and Burch as a replacement alternative that substitutes use of a phylogenetically higher species with a lower one, or perhaps better stated, a later-evolved vertebrate with an earlier-evolved one [7]. An appreciation of the phylogenetic classes of animals and differences between species from one class to another is essential for a rational exploration of animal welfare and ethical issues for biomedical research. Even if there is a capacity for pain as we know it in nonhuman animals, there is good reason to suspect that this "pain potential" is correlated with phylogeny. This is an important ethical consideration, as it has been suggested that "it could be morally appropriate to select animals for scientific use based on their capacities for more or less negative experiences." [3] As previously stated, there is less certainty about the capacity of a species to experience pain as we move from humans to other mammals to lower vertebrates [4]. However, a sentience scale can be construed that parallels evolution, and differences in pain capacity between classes of animals may be supported as "the sentience level of an animal is intimately related to its ability to perceive pain." [4] Adding scientific evidence from comparative neurology may also support a gradation of the capacity for pain among vertebrates.

Comparative neurology of pain. Comparing amphibian and mammalian brains, there are significant differences in both the discriminative and affective pathways of pain transmission [9, 11]. There is no thalamus-to-cortex connection in the frog because a cerebral cortex does not appear until class Reptilia. Even this primordial cortex in reptiles is scant and lacking the complex laminar structure seen in mammals. Amphibians simply have a brain without a cerebral cortex. The phylogeny of the medial pathway correlated with motivational-affective aspects of pain is similar whereby in amphibians the most rostral projection reaches to a diffuse olfactory area with little organization of neurons. In mammals, the most rostral target of this pathway is the highly-organized limbic cortex. Again, the beginning of even a rough laminar organization of the limbic area does not appear until class Reptilia. The amphibian brain does not contain a limbic cortex. Cortical tissue, whether in limbic or cerebral regions, is a highly complex and laminated structure which is a relatively recent development in the evolution of the nervous system. We know from human experience, that decreasing the activity of cortical neurons by anesthesia or surgical lesion results in a loss of the full appreciation of pain [22]. Recent studies using positron imaging techniques also show specific areas of the cortex activated by noxious stimuli in awake humans [20]. For these reasons, there is widespread agreement among various scientific organizations that an intact cortex is needed for the appreciation of pain [1, 8, 21]. It is likely that amphibians, without either cerebral or limbic cortices, have a vastly diminished potential for the appreciation of pain.


To: [Introduction] | Why Do Pain Research | Special Nature of Animal Use in Pain Research | A Case of Comparative Substitution Using Amphibians | Studies of Opioid Analgesia Using Amphibians | Conclusions | Acknowledgments | References

Studies of Opioid Analgesia Using Amphibians

Pezalla first described a method to assess the nociceptive threshold (NT) in frogs using the acetic acid test [5]. The acetic acid test (AAT) to determine the nociceptive threshold in frogs consists of eleven concentrations of acetic acid serially diluted from glacial acetic acid. Nociceptive testing is done by placing, with a Pasteur pipette, a single drop of acid on the dorsal surface of the frog's thigh. Testing begins with the lowest concentration and proceeds with increasing concentrations until the NT is reached. The NT is defined as the lowest concentration of acid that causes the frog to vigorously wipe the treated leg. To prevent tissue damage, the acetic acid is immediately rinsed off with a gentle stream of distilled water once the animal responds, or after 5 seconds if the animal fails to respond. Our results show that using the AAT in amphibians gives a rank order of the relative analgesic potency highly correlated with that found in rodent models after systemic and spinal administration of mu-, delta-, and kappa-selective opioids [10, 15]. These results suggest that the analgesic action of opioid agents in amphibians is predictive of the analgesic effects of opioids seen in humans and other mammals. This fundamental finding also supports use of an amphibian model for the high throughput testing of potential analgesic agents, where lower cost may be an advantage.
To: [Introduction] | Why Do Pain Research | Special Nature of Animal Use in Pain Research | A Case of Comparative Substitution Using Amphibians | Studies of Opioid Analgesia Using Amphibians | Conclusions | Acknowledgments | References

Conclusions

Comparative substitution is a moderate approach to animal replacement alternatives as a whole animal is still used rather than cells or tissue, but amphibians may have considerably less potential for pain than mammalian models currently in use. This is important as whole animals must be used for pain and analgesia research ("cells do not feel pain").

In general, groups promoting the 3Rs of animal welfare for biomedical research have overlooked the immediate welfare gains that may be possible by using comparative substitution as an alternative model. Finally, these studies provide novel data on the efficacy of opioid analgesics in amphibians that may be important for the veterinarian treating amphibians in the clinic.


To: [Introduction] | Why Do Pain Research | Special Nature of Animal Use in Pain Research | A Case of Comparative Substitution Using Amphibians | Studies of Opioid Analgesia Using Amphibians | Conclusions | Acknowledgments | References

Acknowledgments

Research supported by National Institutes of Health (DA07326) and the Whitehall Foundation. Adapted from a talk given at the 2nd World Congress on Alternatives and Animal Use in the Life Sciences, Utrecht, The Netherlands, October, 1996.

The author may be reached at Craig W. Stevens, Ph.D., Dept. of Pharmacology/Physiology, OSU-COM, 1111 West 17th Street, Tulsa, OK 74107-1898 USA, phone: (918) 561-8234, fax: (918) 561-8412, email: scraig@osu-com.okstate.edu


To: [Introduction] | Why Do Pain Research | Special Nature of Animal Use in Pain Research | A Case of Comparative Substitution Using Amphibians | Studies of Opioid Analgesia Using Amphibians | Conclusions | Acknowledgments | References

References

  1. Andrews, E. J., Bennett, B. T., Clark, J. D., Houpt, K. A., Pascoe, P. J., Robinson, G. W., and J. R. Boyce (1993). Report of the AVMA panel on euthanasia. Journal of the American Veterinary Medical Association 202:229-249.

  2. Brenner, G. M., Klopp, A. J., Deason, L. L., and C. W. Stevens (1994). Analgesic potency of alpha adrenergic agents after systemic administration in amphibians. Journal of Pharmacology and Experimental Therapeutics 270: 540-545.

  3. Dresser R. (1989). Ethical and regulatory considerations in the use of cold-blooded vertebrates in biomedical research. In Nonmammalian Animal Models for Biomedical Research, Woodhead, A. D. and K.Vivirito (eds.), CRC Press:Boca Raton, pp. 369-376.

  4. Orlans, F. B. (1993). In the Name of Science: Issues in Responsible Animal Research. Oxford University Press: Oxford 1993.
  5. Pezalla, P. D. (1983). Morphine-induced analgesia and explosive motor behavior in an amphibian. Brain Research 273:297-305.

  6. Pezalla, P. D. and C.W. Stevens (1984). Behavioral effects of morphine, levorphanol, dextrorphan and naloxone in the frog, Rana pipiens. Pharmacology, Biochemistry and Behavior 21: 213-217.

  7. Russell, W. M. S. and R. L. Burch (1959). The Principles of Humane Experimental Technique. Charles C. Thomas, Co.: Sprigfield.

  8. Smith, J. A. and K. M. Boyd (1991). Lives in the Balance. Oxford University Press: Oxford.

  9. Stevens, C.W. (1997). Amphibian models of nociception and pain. In Animal Models of Nociception and Pain, Kavaliers, M. K., Ossenkopp, K. P., and P. R. Sanberg (eds.) R.G. Landes Co.: Austin.

  10. Stevens, C. W. (1996). Relative analgesic potency of mu, delta and kappa opioids after spinal administration in amphibians. Journal of Pharmacology and Experimental Therapeutics 276: 440-448.

  11. Stevens, C. W. (1995). An amphibian model for pain research. Lab Animal 24: 32-36.

  12. Stevens, C. W. (1992). Alternatives to the use of mammals for pain research. Life Sciences 50: 901-912.

  13. Stevens, C. W. and G. M. Brenner (In Press) Spinal administration of adrenergic agents produces analgesia in amphibians. European Journal of Pharmacology.

  14. Stevens, C. W. and K. L. Kirkendall (1993). Time course and magnitude of tolerance to the analgesic effects of systemic morphine in amphibians. Life Sciences 52: PL111-116.

  15. Stevens, C. W., Klopp, A. J., and J. A. Facello (1994). Analgesic potency of mu and kappa opioids after systemic administration in amphibians. Journal of Pharmacology and Experimental Therapeutics 269: 1086-1093.

  16. Stevens, C.W. and P. D. Pezalla (1983). A spinal site mediates opiate analgesia in frogs. Life Sciences 33: 2097-2103.

  17. Stevens, C. W. and P. D. Pezalla (1984). Naloxone blocks the analgesic action of levorphanol but not of dextrorphan in the leopard frog. Brain Research 301: 171-174.

  18. Stevens, C. W., Pezalla, P. D., and T. L. Yaksh (1987). Spinal antinociceptive action of three representative opioid peptides in frogs. Brain Research 402: 201-203.

  19. Stevens, C.W., Sangha, S. A., and B. G. Ogg (1995). Analgesia produced by immobilization stress and an enkephalinase-inhibitor in amphibians. Pharmacology, Biochemistry, and Behavior 51: 675-680.

  20. Talbot, J. D., Marrett, S., Evans, A. C., Meyer, E., Bushnell, M. C., and G. H. Duncan (1991). Multiple representations of pain in human cerebral cortex. Science 251:1355-1358.

  21. Van Sluyters, R. C. (1991). Handbook for the Use of Animals in Neuroscience Research, Society for Neuroscience, Committee on Animals in Research: Washington D.C.

  22. White, J. C. and Sweet, W. H. (1969). Pain and the Neurosurgeon, Charles C. Thomas, Co.: Springfield.

  23. Zimmerman, M. (1983). Ethical guidelines for investigations of experimental pain in conscious animals. Pain 16:109-110.

This article appeared in the Animal Welfare Information Center Newsletter, Volume 8, Number 3/4, Winter 1998

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