LABORATORY ANIMAL MEDICINE AND SCIENCE _ SERIES II

RATS AND MICE: Introduction and Use in Research Part II V-9040

William P. Porter, DVM

Marion Merrell Dow Inc. Cincinnati, Ohio

The Laboratory Animal Medicine and Science - Series II - has been developed by the Autotutorial Committee of the American College of Laboratory Animal Medicine (ACLAM): C. W. McPherson, DVM, Chair; J. E. Harkness; DVM;J. F. Harwell, Jr., DVM; J. M. Linn, DVM; B. J. McGough, BS Medical Communication; A. F. Moreland, DVM; G. L. Van Hoosier, Jr., DVM Instructional development and production assistance provided by Barbara Macfadden. The development of this program is supported by a grant from The Burroughs Wellcome Fund.

Laboratory Animal Medicine and Science Series II is produced by the Health Sciences Center for Educational Resources University of Washington

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AUDIENCE

Veterinary and biomedical students, animal care technicians, and research investigators. RATS AND MICE: Introduction and Use in ResearchPart II is one of a series of autotutorial programs designed to assist in teaching veterinary and other biomedical science students basic information concerning laboratory animal medicine and science. At the conclusion of Part II of this program you should be able to: 1. discuss the advantages and disadvantages of using rodents as research models and give several examples. 2. list several signs of pain or distress in rats or mice. 3. describe preferred analgesics, anesthetics, and methods of euthanasia for rats and mice. Both Part I and Part II of Introduction and Use in Research of rats and mice are designed to stand alone; however, together they form a more complete overview and introduction to the other programs in this series.

GOAL

OBJECTIVES

V-9040 - RATS AND MICE: Introduction and Use in ResearchPart II

1. Series 2. Program title 3. Objectives

Laboratory Animal Medicine and Science Series II Rats and Mice: Introduction and Use in ResearchPart II At the conclusion of this program you should be able to: discuss the advantages and disadvantages of using rats and mice as research models and give several examples. list several signs of pain or distress in rats or mice. describe preferred analgesics, anesthetics, and methods of euthanasia for rats and mice.

4. Section title 5. History of rat use

SUMMARY OF PART I In Part I of this program, the history of the use of rats and mice in research was discussed. The ancestors of the common laboratory rat, Rattus norvegicus, probably followed the lines of human migration throughout the world. Its early domestication was most likely a byproduct of a 19th century sporting event known as rat-baiting. The sport provided an opportunity for inquisitive individuals to tame the more distinctive ratsthe albino, black, and piebald mutants. By the late 1800's, stocks developed in Europe were being used for research purposes in the United States. For example, in the 1890's rats were used in neuroanatomical studies at the University of Chicago. By 1930 there were more than 12 inbred strains of rats established, and rats were accepted widely as standard research animals.

6. History of mouse useThe laboratory strains of mice used today (Mus musculus) are descendants of the European house mouse (Mus domesticus). By the 17th and 18th centuries, mice were being used in studies of anatomy and respiration. In the 19th century, albino and color mutants were generated, and investigations of varietal characteristics and inheritance progressed. By the 1920's, many of the strains of mice used in research today were already established. 7. Part I included Part I also included a discussion of the general characteristics of rats and mice that make them good research animals. These rodents are available from commercial producers. There are large data bases available as a result of years of selective breeding to meet specific research requirements. There are a number of inbred and outbred varieties, as well as many mutant stocks, that are models of human diseases.

8. Section title 9. Introduction

RESEARCH USE Part II of this program will describe specific examples of inbred, outbred, and hybrid rodents used in research. Concerns, techniques, analgesics, anesthetics, and euthanasia will also be covered.

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10. Diabetic mice These mice are both of the 129/J inbred strain, but the larger mouse on the left carries the genotype for diabetes (db/db). Homozygous diabetic mice are recognized by 3 or 4 weeks of age by size difference. They increase in weight rapidly from 3- to 8-weeks of age, reaching a maximum weight of about 45 g. From this point on, their weight declines steadily until death. Obesity, associated with overeating, is accompanied by hyperglycemia. Blood sugar concentrations of 300 mg/dl serum at 4 weeks of age are not uncommon, and by 12 weeks of age, blood sugar levels are usually greater than 500 mg/dl. 11. Dwarf mice These mice are used to study pituitary dwarfism. They are from the inbred strain C3H/HeJ, but the smaller one has a dwarf (dw/dw) genotype which causes deficiency or absence of growth hormone or prolactin or both. The deficiency depresses protein synthesis, a consequence of depressed RNA synthesis.

12. Obese mice Obese mice (ob/ob) were discovered at the Jackson Laboratory in 1949 and are used to study obesity. They are distinguishable from their normal littermates (+/+ and ob/+) at about 4 weeks of age. They gain weight rapidly and reach a maximum weight of about 70 g at 8- to 9-months of age. Their normal littermates reach a maximum weight of about 30 g at 3- to 4-months of age. From the peak of about 70 g, their weight declines slowly until shortly before death. The lifespan of obese mice is about 20 to 24 months, a few months shorter than their normal littermates. Obese mice have a variety of metabolic defects including increased lipogenesis, decreased lipolysis, and obesity itself. All of these can be attributed to hyperphagia and hyperinsulinemia.

13. New Zealand mice

Hybrid mice resulting from breeding New Zealand Black mice with New Zealand White mice develop eye, kidney, and joint disease. These diseases model systemic lupus erythematosus, a condition that primarily affects women of child-bearing age.

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14. Radiation

Mice can be useful in studies of radiobiology, because they tolerate high doses of radiation. Research into the effects of radiation on mice can provide clues on how to avoid problems for people undergoing radiation treatment for cancer. Also, because of their small size, the amount of radiation needed for these studies can be kept at lower levels than if larger animals, such as rabbits, were used.

15. Pneumocystis infection As seen in this lung section, mice and rats can carry the silver-staining micro-organism Pneumocystis sp. in their lungs, yet remain healthy. When rodents are immunodeficient or immunosuppressed with drugs or irradiation, the animals develop pneumocystis pneumonia and usually die. This disease was once rare in man but is now of great importance in humans with AIDS, who often develop pneumocystis pneumonia.

16. Dental lesions One use of rats in dental research is to study carious lesions. When the oral flora of the rat is combined with a cariogenic strain of Streptococcus sp., and the rat is fed a high sucrose diet, the lesions in the enamel of the rats teeth are both macroscopically and microscopically identical to carious lesions of human teeth.

17. Transplantable tumors The tumor types shown here can be transplanted into the rat. Notice that the types have been grouped according to the host stock or strain required for perpetuation of each tumor. The laboratory mouse is the primary animal used in experimental oncology, but rats, because of their size, can provide some significant advantages for this type of study.

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18. Murphy-Sturm tumor The lesion shown here resulted from subcutaneous inoculation of a Wistar albino rat with a Murphy-Sturm lymphosarcoma. This rapidly growing, invasive tumor provides a valuable model for studying the pathogenesis and therapy of tumors.

19. Inhalation chamber When rats are placed intermittently in an inhalation chamber, they may be exposed to various gas or particulate mixtures. The heart and lung lesions that may develop, mimic the lesions found in humans living in various high-risk environments. This is a valuable model that can lead to further understanding of how disease develops, and how it can be prevented or cured in humans.

20. WAG/Rij rat The WAG/Rij rat shown here can be bred to model the development of a serious condition in humans called retinitis pigmentosa. Humans develop the disease early in life, as does this inbred rat.

21. Testicular tumor

Approximately 65% of all male Fischer-344 rats develop a testicular interstitial cell tumor. This tumor develops by 18-to 24-months of age and is associated with atrophy of the secondary sex glands, such as the prostate. There is a similar disease found in man.

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22. Hypertensive rat This image shows one variety of spontaneously hypertensive rat. They develop high blood pressure, sometimes with obesity. The lifespan of the animals that become obese is approximately half that of their non-obese littermates. Hypertension and obesity are serious conditions in humans and have drawn considerable research attention.

23. Rat kidney section This image is a microscopic view of a rat's kidney. The rat kidney has superficial nephrons that can be punctured easily using microscopic techniques. This characteristic makes rats attractive for kidney research, because other animals usually have less accessible nephrons.

24. Section title

BIOMETHODOLOGY

25. Nonspecific illness This image shows the hunched posture and rough hair coat characteristic of a sick mouse. Generally rats and mice behave similarly if distressed or in pain. While it is true that many diseases and conditions produce similar clinical signs in these animals, there are specific etiologies that cause specific problems.

26. Intrasex aggression Male mice housed together may be aggressive and will often fight and mutilate one another. Because of this aggression, male mice should usually be housed separately.

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27. Acute pain signs

Anorexia can sometimes be the earliest sign of health problems or pain in an animal. A decrease in food intake can be transient and not related to pain or discomfort, but it should be considered in determining the condition of the animal. Rats or mice that have had surgery may exhibit signs of pain or discomfort by rubbing or scratching accessible surgical sites, such as the eye or ear. General signs of acute pain or discomfort can include biting or shaking an affected limb or foot, vocalization on movement or palpation, restlessness, lacrimal porphyrin discharge in rats, and increased respiratory rate. Chronic pain or discomfort may generally include the following signs: decreased body weight or reduced weight gain in growing animals, reluctance to move, change in behavior when approached or handled, accumulation of body secretions due to poor grooming, change in bowel or urinary activity, hunched posture, and a rough hair coat. None of the acute or chronic clinical signs is an absolute indication of pain or distress. Experience and professional judgment must be used, along with specific clinical laboratory tests, to determine the cause and severity of the problem.

28. Chronic discomfort

29. Identification methods This image shows an ear punch, one of the methods used for individual identification of mice and rats. Other methods include fur dyes, ear tag, tattooing, micro-chip implantation, and cage cards. Toe clipping is usually considered unacceptable, because suitable alternatives are available.

30. IP injection This image shows the proper restraint of rats and mice during intraperitoneal (IP) injection and anesthesia induction. Restraint is an important factor, not only for the comfort of the animal but for the safety of the person administering anesthesia. An animal that moves suddenly as it is injected could cause an accidental injection of the anesthetist or needless injury to the animal.

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31.

Section title

ANESTHESIA, ANALGESIA, AND EUTHANASIA

32. Drug in syringe Anesthetic drug doses usually differ between rats and mice. It is important to verify the correct dosage for each species, because overdosing these small animals is relatively easy. Injectable anesthetics may be diluted to at least half strength with saline solution to facilitate accurate dose levels and reduce irritation.

33. List of anesthetics

Injectable anesthetics in rats and mice include sodium pentobarbital given intraperitoneally (IP), or ketamine in combination with xylazine or acetylpromazine, given IP or by the intramuscular (IM) route. IM administration of ketamine at high doses can cause muscle necrosis. Hypothermia is used sometimes for brief periods of anesthesia in fetal or newborn animals.

34. Analgesics

Pain relief should be considered, if consistent with study requirements. Injectable analgesics in rats and mice include butorphanol given subcutaneously (SC), meperidine given SC or IM, or pentazocine given SC.

35. Inhalation anesthesia A bell jar or face mask connected with a calibrated vaporizer can be used to induce and maintain adequate anesthesia. The gas anesthetics that can be used include isoflurane, methoxyflurane, or halothane. Ether was used commonly in the past but is now discouraged because of its explosive hazard. Selection of the anesthetic agent and route of administration should be based on the type of experimental procedure that will be performed. Further information on anesthesia is available in programV-9054, LABORATORY ANIMALS: Surgery and Anesthesia in Rodents.

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36. Euthanasia methods Rats and mice can be euthanatized by several methods including injection with barbiturates or other suitable euthanasia solution; inhalation of carbon dioxide, methoxyflurane, or halothane; and decapitation or cervical dislocation with or without sedation, depending on the nature of the study. Because large numbers of these animals are often on study at one time, euthanatizing several animals at a time could be necessary. In such cases, crowding of animals should be avoided. 37. Cervical dislocation Physical methods of euthanasia, such as cervical dislocation shown in this image, can be performed with or without sedation or anesthesia depending on study requirements. This method should be used only on small or young animals, and when the nature of the study precludes the use of chemicals. It should also be scientifically justified and approved by the Institutional Animal Care and Use Committee.

38. Mouse in desiccator jar This image shows a mouse in an inhalation chamber. At no time should animals have direct contact with chemical anesthetic or euthanasia substances, such as liquid methoxyflurane or halothane. To prevent epithelial irritation or burns, there should be no direct contact with dry ice, which is sometimes used to generate carbon dioxide.

39. Confirmation of death Death of an animal after euthanasia administration should be confirmed before carcass disposal. This can be done by checking for respiration a few minutes after a procedure is completed, by exsanguination, or by opening the chest cavity to collapse the lungs. The 1993 report of the American Veterinary Medical Association Panel on Euthanasia provides more information on this subject (1).

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40. Conclusion

This image concludes this autotutorial session on the introduction and use in research of rats and mice. There are seven programs in this series: V-9039 RATS AND MICE: Introduction and Use in ResearchPart I V-9040 RATS AND MICE: Introduction and Use in ResearchPart II V-9041 RATS AND MICE: Biology V-9042 RATS AND MICE: Care and Management V-9043 RATS AND MICE: Bacterial and Mycotic Diseases V-9044 RATS AND MICE: Viral Diseases V-9045 RATS AND MICE: Parasitic Diseases

41. ACLAM credits This program was developed for the American College of Laboratory Animal Medicine. C. W. McPherson, DVM Chair J. E. Harkness, DVM J. F. Harwell, Jr., DVM J. M. Linn, DVM B. J. McGough, BS A. F. Moreland, DVM G. L. Van Hoosier, Jr., DVM Instructional development, editing, and production management provided by Barbara Macfadden. Guide production supported by Pamela Young. The development of this program was supported by a grant from The Burroughs Wellcome Fund.

42. HSCER credits Produced by the Health Sciences Center for Educational Resources University of Washington Seattle WA 98195 (206) 685-1156 Fax: (206) 543-8051 2000

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ACKNOWLEDGMENTS Images 10 and 11 are provided courtesy of The Jackson Laboratory, Bar Harbor, Maine. Image 12 is provided courtesy of J. Russell Lindsey, DVM, University of Alabama-Birmingham. Image 15 is provided courtesy of Charles W. McPherson, DVM, North Carolina State University. Image 20 is provided courtesy of Gail C. Wolz, University of Washington, Seattle. Image 22 is provided courtesy of William P. Porter, DVM, Marion Merrell Dow Inc., Cincinnati, Ohio.

REFERENCES 1. American Veterinary Medical Association. 1993. Report of the AVMA panel on euthanasia. J. Am. Vet. Med. Assoc. 202(2):229-249.