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8 Rodents that Require Special Consideration Rodents with a wide variety of valuable genetic characteristics are avail- able for use in many kinds of research (Altman and Katz, 1979a, 1979b; Festing, 1993; Festing and Greenhouse, 1992; Hansen et al., 1981; Hedrich and Adams, 19901. Most are easily maintained with the husbandry techniques discussed in Chapter 5. However, some important research models, especially those with deleterious mutations, require special care. Some such as mice that carry the homozygous mutation scid (severe combined immune deficiency), some strains of mice that carry the homozygous mutation nu (nude), and rodents exposed to sublethal irradiation are so severely immunodeficient that contact with infectious agents of even low pathogenicity can cause severe illness and death, and they require isolation for survival (NRC, 1989~. Others have specific requirements for the presentation of food and water; for ex- ample, food pellets must be placed on the cage floors and longer than normal sipper tubes are necessary for rodents with mutations that cause dwarfing, and soft diets are essential for mice and rats with mutations in which the incisors fail to erupt (Marks, 19871. Many mutants are subfertile or sterile and require special breeding techniques to maintain the mutation. A detailed description of the unique husbandry and breeding requirements for each model is beyond the scope of this book. Mating strategies for propa- gating lethal, sterile, or deleterious mutations have been described (Green, 1981~. Those wishing to use mutant rodents should discuss with the investiga- tor or company providing the animals whether there are special requirements for the animals' care and breeding. This chapter will address selected research 121
122 RODENTS: LABORATORY ANIMAL MANAGEMENT models: immunodeficient rodents, wild rodents, rodents used for studying aging, mouse and rat models for type I (insulin-dependent) diabetes mellitus, and transgenic mice. Those models are relatively commonly used in research, and information on their husbandry is often difficult to find. IMMUNODEFICIENT RODENTS Rodents whose immune systems have been altered through spontaneous mutation, transgenic manipulation, or the application of immunosuppressive drugs or other treatments have long been useful models in biomedical re- search. However, the immunologic deficiencies that make these animals use- ful as models often render them susceptible to a host of opportunistic and adventitious infectious agents that would produce few or no effects in immu- nologically competent animals (Powles et al., 1992; Soulez et al., 1991~. The recommendations in this report that cover various rodent species generally apply to immunologically compromised rodents, but much more stringent housing conditions are often required to ensure the health of immunodeficient rodents. Husbandry In general, the cages or other implements used to house immunodeficient rodents should be capable of being adequately disinfected or sterilized on a regular basis. The housing systems should be capable of eliminating airborne contamination of the animals and should be capable of being manipulated without exposing the animals to microbiologic contamination during experi- mentation and routine husbandry procedures. In determining housing and husbandry requirements for maintaining immunodeficient rodents, it is impor- tant to consider the effects of various opportunistic and adventitious microor- ganisms on the type of research being conducted. The length of the study and the research goals will influence the attention to detail needed to prevent infection with such organisms. Maintaining animals in an axenic or microbio- logically associated (defined-flora) state might involve a level of effort that is too great and techniques that are too complex for most experimental studies. Plastic Cages with Filter Tops This housing system consists of a shoebox cage usually constructed of transparent autoclavable plastic and a separate filter top a plastic cap with a removable filtration surface in the top. The cap and cage fit together snugly but do not necessarily form a perfect seal. A stainless-steel wire-bar top keeps animals from gaining access to the filter top and provides a food hopper and a holder for a water bottle. An opaque cage can be used, but a transparent cage facilitates routine animal observation without the need to open the cage except
RODENTS THAT REQUIRE SPECIAL CONSIDERATION for feeding and watering, sanitation, and experimentation. 123 Cages and filter tops and all food, water, and bedding used in those cages should be sterilized. All changing and manipulation of animals should be done in a laminar- flow work station using aseptic technique. Sterile gloves or disinfected for- ceps should be used to manipulate animals in any individual cage, and all experimental manipulations should be done so as to minimize or eliminate contamination of the animals. The successful maintenance of animals with this housing system depends directly on rigid adherence to aseptic technique in all aspects of animal and cage manipulation. Although the initial purchase cost of this housing system might seem relatively low compared with that of other systems for housing immunodeficient rodents, the requirement for lami- nar-flow change stations, sterile supplies, and other operating expenses leads to a substantial continuing cost. Moreover, only minimal mechanical safe- guards are built into this system, and success depends absolutely on technique. A major drawback to using plastic cages with filter tops is that there is a low rate of air exchange between the cage and the room. As a result, bedding might have to be changed more frequently to minimize the buildup of toxic wastes and gases and keep relative humidity appropriately low. Individually Ventilated Plastic Cages with Filter Tops This housing system uses plastic cages with filter tops that are constructed and maintained like those previously described. However, an air supply has been introduced into each cage with a special coupling device similar in ap- pearance to the fittings used for automatic watering. Air is supplied to a cage under positive pressure and is exhausted through the filter top. Other ventila- tion options with respect to positive and negative pressure, as well as a sepa- rate exhaust, are also available. Usually, the air supplied to these cages is filtered with a high-efficiency particulate air (HEPA) filter. This system has advantages over the nonventilated plastic cages, but its principal disadvantage is the potential for contamination of the fittings that are used to introduce air into the cages. Rigorous attention must be paid to disinfection of these fit- tings. The efficiency of this system in protecting immunodeficient animals from infectious agents has not been extensively evaluated. Isolators Large isolators capable of housing many rodent cages are commercially available. As discussed elsewhere in this report, isolators are ideal for excluding microorganisms in that they rely very little on individual tech- nique for many husbandry procedures or experimental manipulations. Tra- ditionally, they have been used for housing axenic or microbiologically associated animals. Many varieties of isolators are available; the most common are those made of a flexible bag of vinyl or other plastic material,
24 RODENTS: LABORATORYANIMAL MANAGEMENT such as polyurethane. Modern isolators are relatively easy to use and pro- vide investigators and animal-care technicians with easy access to the ani- mals. Special precautions are not needed, because all manipulation is done through built-in glove sleeves with attached gloves. All supplies provided to the isolator are sterilized and are introduced through a port; a chemical sterilization and disinfection procedure is used to decontaminate the outside of the items that have been previously sterilized and wrapped with plastic or other materials that can withstand chemical sterilization or disinfection. Air into and out of the isolator is usually highly filtered. As opposed to plastic cages with filter tops, the isolator offers an advantage in health assessment, in that a large number of animals are maintained as a single biologic unit. Isolators made of rigid plastic with a flexible front offer additional advantages, such as integrated racking, individual lighting, lower operating air pressures, and conservation of space. Recent advances in construction coupled with the availability of vacuum- packed and irradiated supplies have made isolators for housing immuno- logically compromised animals a cost-competitive alternative to cages with plastic filter tops. HEPA-Filtered Airflow Systems These systems have a variety of forms, including modular chambers, hoods, and racks that are designed to hold cages under a positive flow of HEPA-filtered air. In some instances, plastic cages with filter tops have been used in laminar airflow racks that supply a steady stream of HEPA- filtered air across the cage tops to facilitate air diffusion through the filters. The design of such racks usually involves a blower that pushes air across a HEPA filter and then into a large space (or plenum) that contains thousands of small holes. The holes are designed to permit air to be blown across shelves on which cages are placed. Because many cages must fit on the shelves, there is considerable eddying or turbulence of air across the tops of the cages. Once the cages are pulled forward 10-20 cm beyond the lip of a shelf, the air no longer flows laminarly and mixes with room air. Another system consists of a flexible-film enclosure in which HEPA-filtered air is supplied under positive pressure to a standard rack or group of racks con- taining filter-topped cages. For both systems, all manipulations must be made in a laminar-flow work station using aseptic technique. Environmental Considerations Immunodeficient rodents have been successfully maintained at rec- ommended room temperatures for rodents (NRC, 1996 et seq.~. Several theoretical considerations suggest that some immunodeficient rodents, specifically those lacking hair or thyroid glands, might require a higher
RODENTS THAT REQUIRE SPECIAL CONSIDERATION 125 ambient temperature because of hypothyroidism and poorly developed brown adipose tissue, which reduce the capability for nonshivering thermogenesis (Pierpaoli and Besedovsky, 1975; Weihe, 1984~. In practice, such temperatures are not necessary and in fact can be detrimental be- cause they tend to create husbandry problems, including increased de- composition of feed and bedding, increased rate of growth of environ- mental bacteria, and an uncomfortable working environment for animal-care personnel. In addition, because housing of immunocompromised ani- mals generally requires systems that restrict airflow and heat transfer, temperatures in the animal cages tend to be higher than ambient tem- perature; therefore, increasing the room temperatures is generally not necessary. Humidity and ventilation should be consistent with recommendations in the Guide (NRC, 1996 et seq.~. It is important to remember that many of the containment systems result in increased relative humidity and restrict ventilation. Therefore, animal density, bedding-change frequency, and the relative humidity of incoming air should be adjusted to compensate for some of these differences. Food and Bedding Food and bedding for immunocompromised animals should be steril- ized or pasteurized to eliminate vegetative organisms. Depending on the method of sterilization selected, fortification of feed with vitamins might be required. Steam sterilization can drastically reduce concentrations of some vitamins and can accelerate the decomposition of some vitamins during storage. Other treatments, such as irradiation, result in much less destruc- tion of these nutrients and so might not require the same degree of fortifica- tion of feed before or after sterilization. Adequate validation of the steril- ization process is essential to ensure that food or bedding does not serve as a source of contamination. Water The water supplied to immunodeficient animals must be free of micro- biologic contamination. Sterilization of water is the only sure method of eliminating such contamination. Sterilization can be accomplished by heat treatment, zonation, or filtration. All those processes must be adequately controlled and validated. Other water treatments have been advocated for use with immunocompromised animals, including acidification, chlorina- tion, chloramination, and the use of antibiotics and vitamins. The principal purpose of adding treatment materials to water is to reduce bacterial growth and hence the likelihood of cross contamination in case bacteria are intro
126 RODENTS: LABORATORYANIMAL MANAGEMENT duced into the water supply The treatments are not without effects, which can include alteration of bacterial flora, alterations in macrophage and lym- phocyte function, reduction in water consumption, and exposure to chlori- natedhydrocarbons(Fidler, 1977;Halletal., 1980;Hermanetal., 1982; McPherson, 1963; Reed and Jutila, 19729. In general, the use of the treat- ments is not an adequate substitute for sterilization of water and should be used only as an adjunct. Health Monitoring Many immunodeficient rodents are susceptible to a greater range and incidence of diseases caused by microorganisms than are immunocompetent animals. The lack of a completely functioning immune system often results in more dramatic clinical signs and pathologic changes than would be seen in immunocompetent animals. Because some immunodeficient animals of- ten lack the ability to produce antibodies in the presence of microorgan- isms, serology is often not useful for diagnosis. Screening for such agents might require the use of immunocompetent sentinel animals of the appropri- ate microbiologic status. Most commonly, soiled bedding is used as a means of exposing sentinel animals to the immunocompromised animals, and a period of 4-6 weeks of exposure is often required before samples can be taken. Sentinels must be housed under the same environmental condi- tions and microbiologic barriers as the immunocompromised animals. Health monitoring of animals maintained in individual plastic cages with filter tops is complicated by the potential for contamination of individual cages, as opposed to large groups of cages, with a particular microorganism. Be- cause frequent screening of every cage is not economically feasible, statisti- cal schemes for sampling or batching soiled bedding for exposure of senti- nel animals is often required. That is less of a problem with the use of isolators in which large numbers of cages are kept in the same microbio- logic space. Purchase of animals from commercial sources or transfer of animals from other institutions entails some risk with respect to immunocompromised animals. Health status can be compromised during packing, transport, un- packing, and housing of animals. It is important to provide adequate quar- antine and stabilization time to allow assessment of the health status of these animals before they are used in experimental procedures. Appropriate precautions should be taken to disinfect the outside of transport containers and to examine them for integrity. Specialized containers have been devel- oped for transport of immunocompromised rodents and should be used whenever possible.
RODENTS THAT REQUIRE SPECIAL CONSIDERATION WILD RODENTS 127 A large number of rodent species have been maintained and bred in a laboratory environment. Wild rodents are used in many fields of research, including genetics, reproduction, immunology, aging, and comparative physio- logy and behavior. Hibernating rodents, such as woodchucks (Ma rmota monax) and 13-lined ground squirrels (Spermophilus tridecemlineatus), are used to study control of appetite and food consumption, control of endo- crine function, and other physiologic changes associated with hibernation. Woodchucks are also used as models to study viral hepatitis and virus induced carcinoma of the liver. Wild rodents can be obtained by trapping or, in a few instances, from investigators who are maintaining them in the laboratory. Trapping is the simplest way to acquire wild rodents. However, a collector's permit is re- quired in most states, and it is also important to confirm that the species to be trapped, as well as other species in the trapping area, are not threatened or endangered. It is best to begin trapping with an experienced mammalogist. A search of the literature will locate investigators who maintain feral rodents in a laboratory environment; however, these scientists usually do not maintain enough animals to permit distribution of more than a few. Colonies of wild rodents are listed in the International Index of Laboratory Animals (Festing, 1993), in Annotated Bibliography on Uncommonly Used Laboratory Animals: Mammals (Fine et al., 1986), and in the Institute of Laboratory Resources (ILAR) Animal Models and Genetics Stocks Data Base (contact: ILAR, 2101 Constitution Avenue, Washington, DC 20418; telephone, 1-202-334-2590; fax, 1-202-334-1687; URL: http://www2.nas.edu/ ilarhome/~. Several species of the genera Mus and Peromyscus are more widely used and are available from laboratory-bred sources. Hazards Wild-trapped rodents commonly carry pathogens and parasites that are usually not found in or have been eliminated from animal facilities; there- fore, appropriate precautions must be taken to prevent disease transmission between feral and laboratory stocks (see Chapter 63. The primary hazard to personnel is getting bitten. Personnel should always wear protective gloves when handling wild rodents. Mice can be handled with cotton gloves (Dewsbury, 1984) or can be moved from place to place in a tall, thin bottle (Sage, 19811. Metal meat-cutter's gloves can be worn under leather gloves for handling larger, more powerful species, such as black rats (Rattus rattus) (Dewsbury, 19841. Elbow-length protection, such as leather gloves and gauntlets, should be worn for handling woodchucks because the animals can turn rapidly and bite the inside of the handler's forearm.
128 RODENTS: LABORATORYANIMAL MANAGEMENT Wild rodents can carry zoonotic diseases, such as leptospirosis and lymphocytic choriomeningitis, that are not usually encountered in labora- tory-bred rodents (Redfern and Rowe, 19761. Personnel should be offered immunization for tetanus, and anyone that is bitten should receive prompt medical attention. Wild-caught mastomys tPraomys (Mastomys) natalensis] cannot be imported into the United States, because it is a host for the arenavirus that causes the highly fatal Lassa fever. Care and Breeding Many small species can be housed in standard mouse and rat cages (Boice, 1971; Dewsbury, 1974a); solid-bottom cages with wood shavings or other bedding are preferred (Dewsbury, 1984~. Most small wild rodents are much quicker than domesticated rodents and can easily escape if the han- dler is not careful. It is advisable to open cages inside a larger container, such as a tub or deep box, to avoid escapes (Dewsbury, 1984; Sage, 19811. Most species do well if given ad libitum access to water and standard rodent diets; however, voles do better on rabbit diets (Dewsbury, 1984; Fine et al. 1986~. General guidelines for caring for wild rodents have been published (CCAC, 1984; Redfern and Rowe, 19761. Fine et al. (1986) have summa- rized and provided references for laboratory care and breeding of kangaroo rats (Dipodomys spp.~; grasshopper mice (Onychomys spp.~; dwarf, Sibe- rian, or Djungarian hamsters (Phodopus sungorus); Chinese hamsters (Cricetulus barabensis, also called C. griseus or C. barabens~s griseus); common, black- bellied, or European hamsters (Cricetus cricetus); white-tailed rats (Mystromys albicaudatus), fat sand rats (Psammomys obesus), voles (Microtus spp.), four-striped grass mice (Rhabdomys pumilio), and degus (Octodon degus). Guidelines on laboratory maintenance of hystricomorph (Rowlands and Weir, 1974; Weir, 1967, 1976) and heteromyid (Eisenberg, 1976) rodents have been published. Mammalogists and other investigators experienced in working with specific species are also excellent sources of information. Breeding of many wild species is similar to that of domesticated ro- dents. Some (e.g., voles and deer mice) breed almost as well in captivity as do domesticated species (Dewsbury, 19841. Others (e.g., four-striped grass mice) require special conditions (Dewsbury, 1974b; Dewsbury and Dawson, 1979~. A few investigators have reported that breeding of wild Mus species is difficult unless running wheels are provided; exercise (up to 10-15 miles/ day) apparently causes females to come into estrus and begin a normal breeding cycle (Andervont and Dunn, 1962; Schneider, 1946~. Others have not had this problem (Sage, 1981~. Pheromones are extremely important in the reproduction of some wild rodents; too frequent bedding changes pre- clude successful reproduction. A nesting enclosure might be appropriate and should be constructed of a durable material that is easily sanitized, such
RODENTS THAT REQUIRE SPECIAL CONSIDERATION 129 as plastic or corrosion-resistant metal. Nesting material might improve neonatal survival. Peromyscus Peromyscus maniculatus (the deer mouse) and P. Ieucopus (the white- footed mouse) can be maintained with the same husbandry procedures as laboratory mice. A maximum of seven can be housed in 7 x 10 inch plastic cages. Standard rodent feed and water should be give ad libitum. Rabbit or guinea pig feed should not be used, nor should such supplements as fresh vegetables, raisins, and sunflower seeds. Except for breeding, sexes should be housed separately. Peromyscus are reasonably cold-tolerant; the sug- gested temperature is 22-25°C (71.6-77.0°F), and the ambient temperature should not exceed 33°C (91.4°F). For breeding, single male-female pairs are formed at the age of about 90 days and remain together throughout life. _~J ~ ~ ^ ~_~^A_^ ~ ~ The estrous cycle is 5 days (Clark, 19841. Females caged alone or with other females will not come into estrus. The average reproductive life of Peromyscus is 18-36 months. Females should be checked regularly for pregnancies. Copulatory plugs are not a reliable indication of mating, because they are inconspicuous. Light- ing is very important in breeding. A 16:8-hour light:dark ratio is generally satisfactory. Continuous light will produce anestrus, and breeding difficul- ties can sometimes be overcome by reducing the light cycle to a light:dark ratio of 12:12 hours and gradually increasing it to 16:8 over a 3-week period (W. D. Dawson, Peromyscus Stock Center, unpublished). Introduc- tion of a strange male into a cage with a pregnant female can block the pregnancy (Bronson and Eleftheriou, 1963~. Gestation is 22 days, except in lactating females, in which it is delayed by 4-5 days. Females enter post- partum estrus about 12 hours after delivery and then remate; therefore, serial litters are produced at 26- to 27-day intervals. Litter size is usually three to six and rarely exceeds eight. Males provide some of the care for the young. Additional information on the care and breeding of Peromyscus can be obtained from the Peromyscus Stock Center, Department of Biology, University of South Carolina, Columbia, SC 29208 (telephone, 803-777- 3107; fax, 803-777-4002~. - Woodchucks Woodchucks (Marmota monox) have been successfully housed indoors in standard cat, dog, or rabbit cages (Snyder, 1985; Young and Sims, 1979) and outdoors in pens or runs (Albert et al., 1976). Enclosures must be carefully secured because a woodchuck can squeeze through any hole large enough to admit its head (Young and Sims, 1979). Each animal should be
30 RODENTS: LABORATORY ANIMAL MANAGEMENT provided with a nesting box and nesting material, especially if it is housed under conditions that will induce hibernation, for example, in a cold room or, in a cold climate, outdoors in an unheated enclosure. Very thin wood- chucks will not survive hibernation (Young and Sims, 1979~. Usually, adult females are housed in small groups, and males are housed individually except during breeding season. However, young males and females can be kept together through their first year (Young and Sims, 1979~. Food and water should be made available ad libitum. Water should be provided in heavy porcelain bowls. Standard bottles and sipper tubes are not satisfac- tory, because the animals grip the tubes in their teeth and shake them until they are dislodged from the bottles (Snyder, 1985; Young and Sims, 19791. Woodchucks do well on commercial rabbit diet (Young and Sims, 19791. AGING COHORTS Mice and rats have been favored by mammalian gerontologists as ex- perimental models because of their relatively short and well-defined life spans, small size, comparatively low cost, and the large and growing store of information on their genetics, reproductive biology, physiology, biochemistry, endocrinology, neurobiology, pathology, microbiology, and behavior. However, the term comparatively low cost is used advisedly. The true cost in 1994 of producing one 24-month-old rat was approximately $200 and a similarly aged mouse $95; the cost for producing one 36-month-old rat was approxi- mately $350 and a similarly aged mouse $175 (Dewitt Hazzard, National Institute on Aging, National Institutes of Health, Bethesda, Maryland, un- published). The cost to investigators is slightly more than half that amount because production is subsidized by the National Institute on Aging (NIA). A problem faced by investigators who use aged animals is periodic short- ages in older cohorts of some strains. General Considerations Strictly speaking, aging can refer to all changes in structure and function of an organism from birth to death; however, mammalian gerontologists gen- erally confine their experiments to alterations that occur after the onset of sexual maturity and the transition from the juvenile to the young adult pheno- type. In sampling for some measure of aging or accruing pathologic condi- tions, 6-month-old animals will usually provide a normal baseline, and sam- pling should be carried out at 6-month intervals. Many investigators consider a 24-month-old rodent to be "old"; however, age-related changes in a number of characteristics are often more pronounced in still older animals. The mean life span (MnLS) of ad libitum-fed (AL-fed), hybrid strains of specific-pathogen-free (SPF) mice or rats is often around 30 months,
RODENTS THAT REQUIRE SPECIAL CONSIDERATION 131 whereas that for calorically restricted (CR) animals, depending on the regi- men used, can be 30 percent longer (see Figures 8.1 and 8.2~. Because caloric restriction retards or eliminates common forms of chronic renal dis- ease and a variety of neoplasms, some gerontologists believe that such nutritional management should be the norm. Comparative changes in AL- fed versus CR rodents are increasingly used to test the validity of putative biologic markers of aging rates. Survivorship in any colony used for gerontologic research should be de- termined repeatedly. Survival curves for SPF mice and rats should exhibit a classic "rectangularization pattern," that is, a survival curve should nearly parallel the X axis close to the 100-percent survival level for a prolonged period and then decline sharply as the population nears the species' maximum life span (MxLS), which is defined as the age at which only 10 percent of the animals are surviving. A linear survival curve indicates a problem in the population (e.g., exposure to infectious disease). Patterns of age-related pa- thology within a colony should be repeatedly evaluated through systematic sampling and necropsy of cohorts of various ages (including histologic exami- nation of the major organs). Any animal euthanatized during the course of a study on aging should be necropsied to determine whether the cause of death, such as a specific lesion or neoplasm, could seriously affect the interpretation of the experimental data. For example, the occurrence of lymphoma involving primarily the spleen of old mice of some strains not only decreases survival, but might cause death before other expected findings can occur; this limits the value of these strains in some studies of age-related immunology. A good deal of information is now available on the pathology of aging cohorts of com- monly used laboratory mice and rats (Altman, 1985; Bronson, 1990; Burek, 1978; Myers, 1978; Wolf et al., 1988~. Laboratory Mice There are obvious advantages to using genetically defined strains for research on aging. Inbred or F1 hybrid strains provide a reproducible gene pool, and so permit a more rigorous evaluation of environmental variables, such as caloric restriction. However, in some circumstances, such as longi- tudinal studies with markers of aging or searches for longevity-assurance genes, the widest possible allelic variability might be desired. For those purposes, systematically outbred animals might suffice, although in the de- velopment of such lines, including so-called Swiss mice, the tendency to select breeding pairs for docility and breeding efficiency has resulted in a loss of genetic heterogeneity. An alternative approach is to develop an 8- or 16-way cross between established inbred lines (van Abeelen et al., 1989~. Recombinant inbred mice can also be useful for aging research because they provide a reassortment of linked parental genes (see Chapter 31. Re
32 RODENTS: LABORATORY ANIMAL MANAGEMENT combinant congenic strains are of special interest for the analysis of poly- genic traits (Demant and Hart, 1986; van Zutphen et al., 1991) because they contain a small fraction of the genome of a genetically defined donor line against a genetic background derived from another genetically well-defined strain. For a discussion of the specific uses and relative values of inbred, congenic, recombinant inbred, and nongenetically defined populations, see Gill (19801. Eight SPF mouse strains, commonly used for gerontologic studies are available from the NIA: inbred strains A/HeNNia, BALB/cNNia, CBA/ CaHNNia, C57BL/6NNia, and DBA/2NNia and hybrid strains BALB/cNNia x C57BL/6NNia F1 (CB6F1), C57BL/6NNia x C3H/NNia F1 (B6C3F1), and C57BL/6NNia x DBA/2NNia F1 (B6D2F1~. Crl:SW outbred stock is available commercially. Nude mice have also been suggested for gerontologic research (Masoro, 1990), but they are not available from NIA. By using mouse stocks obtained from NIA for research on aging, an investigator avoids changes in genetic characteristics and phenotypes caused by genetic drift in animals from disparate sources (see Chapter 31. An advantage to using well-studied strains is that historical baseline measures are available for comparison, including characteristic age-related pathologic conditions that might complicate the research (see Hazzard and Soban, 1989, 1991, for bibliographies). Life tables for most mouse strains have been published and are summarized by Abbey (1979), and Masoro (1990) presents accumulated data from several sources (see also Green and Witham, 19914. MnLS and MxLS are required in most cases as background data when choosing a strain. More extensive survival data can be obtained from survival curves like those compiled for the SPF colonies of aging NIA mice maintained at the Division of Veterinary Services, National Center for Toxicological Re- search (NCTR) in Jefferson, Arkansas. An example of such a curve for B6D2F1 (AL-fed versus CR) is presented in Figure 8.1. A group of related sublines derived from AKR mice and known as SAM (senescence-accelerated mice) have also been developed. SAM mice display multiple pathologic conditions, have an MnLS of as little as 200 days, and have an MxLS of as little as 290 days. They respond to caloric restriction in the same manner as do other strains of mice (Takeda et al., 1981; Umezawa et al., 19901. Rats (Rattus norvegicus) Four strains are available from NIA: inbred strains BN/RijNia (Brown Norway) and F344/NNia (Fischer 344) and hybrid strains BN/RijNia x F344/ NNia F1 (BNFF1) and F344/NNia x BN/RijNia F1 (FBNF1~. Inbred strains BUF/N (Buffalo) and LEW (Lewis) and outbred stocks LE (Long Evans), SD (Sprague Dawley), and WI (Wistar) have also been used in research on
RODENTS THAT REQUIRE SPECIAL CONSIDERATION 1.0- 0.8- am. . _ ~ 0.6- En o ._ - 0.4- Q o 0.2- O ' ~ % .. , :, ~L, L . ~ _ . . " 1 '- 1 'a.,: .... - .'' 2...,~ : ~ : L .. , 1 1 1 , 1 1 1 l 1 1 1 ' 1 1 1 0 20 40 60 80 100 120 140 160 180 200 220 Time (weeks) 133 240 FIGURE 8.1 Survival of male and female C57BL/6NNia x DBA/2NNia F1 (B6D2F1) mice reared under monitored SPF conditions. Studies conducted for the National Institute on Aging by the Division of Veterinary Services, National Center for Toxico- logical Research, Jefferson, Arkansas. Curves are shown for both AL-fed and CR mice: , AL-fed males; . ., AL-fed females; , CR males; , CR females. Caloric intake for CR mice was 60 percent of that for AL-fed mice. Calories were reduced gradually between 12 and 16 weeks of age and then continued at reduced levels for the remainder of the life span. All mice were individually housed. aging. These are available commercially as young animals but seldom as old animals. Life tables are available for each of those stocks and strains (Hoffman, 1979; Masoro, 1990~. Although rats were previously believed to have longer life spans than mice, recent studies indicate that, the life spans of rats and mice are similar (Table 8.1~. Rats' larger size might make them more useful than mice for some studies of aging, such as those involving surgery, and rats are widely used in studies on the neurobiology of aging. As do mice, aging cohorts of rats exhibit an increased prevalence of various neoplasms. The prevalence of specific kinds of neoplasms varies among strains. Infectious diseases, including a chronic respiratory complex associated with Mycoplasma pulmonis, can also affect life span. The incidence of M. pulmonis in rats has been found to be 38 percent in conventionally housed colonies and O percent in SPF colonies (NRC, 1991~. Thus, cesarean derivation and barrier mainte- nance can eliminate M. pulmonis associated with chronic respiratory disease of rats. Survival curves (AL-fed versus CR) for FBNF1 rats reared under such conditions at NCTR are presented in Figure 8.2.
134 RODENTS: LABORATORYANIMAL MANAGEMENT TABLE 8.1 Mortality for Selected Strains of Mice and Rats Fed Ad Libitum Age, weeks Females Males Strain 50% 90~o 50% 90% Mortality Mortality Mortality Mortality Mice C57BL/6NNia 117 143 120 141 DBA/9NNia 77 123 88 126 C57BL/6NNia x DBA/2NNia F1 (B6D2F1) 128 152 138 171 C57BL/6NNia x C3H/NNia F1 (B6C3F1) 132 158 140 177 Rats F344/NNia 116 144 103 121 BN/RijNia 133 157 129 155 F344/NNia x BN/RijNia F1 137 166 146 171 SOURCE: Data on National Institute on Aging colonies from the Division of Veterinary Services, National Center for Toxicological Research, Jefferson, Arkansas. 0.R- . _ 3 0.6 O _ _ ._ .- 0.4- Q o CL 0.2- O - a . .~. , ... . I ~I ~I ~I ~I ~I ~ I I I ~I I I ~I ~I -r 1 0 20 40 60 80 100 120 140 160 180 200 220 240 Time (weeks) FIGURE 8.2 Survival of male and female F344/NNia x BN/RijNia F1 (FBNF1) rats reared under monitored SPF conditions. Studies conducted for the National Institute on Aging by the Division of Veterinary Services, National Center for Toxicological Research. Curves are shown for both AL-fed and CR rats:- , AL-fed males; AL-fed females; , CR males; , CR females. Caloric intake for CR rats was 60 percent of that for AL-fed rats. Calories were reduced gradually between 12 and 16 weeks of age and then continued at reduced level for remainder of life span. All rats were individually housed. . .
RODENTS THAT REQUIRE SPECIAL CONSIDERATION Husbandry 135 There is evidence of an age-related decline in immune response (Miller, 1991), therefore, maintenance of an SPF microbiologic status, under clearly defined and regularly monitored conditions, is a requirement for an aging . ~,- . . . , Be, ~ ~ colony. evince and rats In an aging colony can be housed in groups (usually four to five animals per cage) or individually. The latter is necessary for both test (CR) and control (AL-fed) animals in caloric-restriction studies. In some colonies, an exercise device, such as a wheel, is provided. The results of studies on whether group housing or exercise facilitation extend MnLS or MxLS vary (slough, 1991; Holloszy and Schechtman, 1991; Masoro, 1991; Menich and Baron, 1984; Skalicky et al., 1984~. A complication of croup housing occurs as the old animals begin to die. When that occurs, cages no longer have identical conditions; some contain several animals and others contain only one or two animals. Another complication of group housing, especially among males, is the fighting and threat stress that oc- curs between animals when dominance is being asserted. The effect of such stress can substantially affect the results of studies on survival, metabolism, and behavior. If males are to be group-housed, they should be grouped immediately after weaning. In some strains, however, this will not prevent fighting. In some instances, the death of one animal in a cage will be followed by the deaths of the rest of the animals in that cage; whether this is caused by an opportunistic pathogen or by the stress of the first animal's death is not clear. an- --r ~-= on Conversely, individual housing is probably stressful initially and might promote inactivity. Thus, the choice of a housing plan depends on the sex and strain of the experimental animals and on the ex- perimental protocol. Room lighting is especially important in gerontologic research in which performance is measured. Because of the retinal damage that can be caused in albino rodents by exposure to moderately bright light (see Chapter 5), placement of individual cages in relation to the lighting source could influ- ence performance over time. An additional consideration is the light:dark cycle. When CR animals are being compared with AL-fed controls, it is desirable to regulate the light cycle so that both groups will begin eating simultaneously, and activity, cell division, hormone concentrations, and other characteristics will be measured in both groups at similar times on the blood-glucose and -insulin curves. Mice and rats are essentially nocturnal, and AL-fed animals naturally begin feeding shortly after the dark cycle begins. CR animals, in contrast, begin to eat immediately after they are fed, which is usually during the light cycle, and consume most of their food quickly. Both sets of animals can be induced to eat at the same time by reversing the light:dark cycle so that the animal room is dark during the workday. If the light:dark cycle is reversed, the illumination used in the room during the workday should not be visible to the animals.
36 RODENTS: LABORATORY ANIMAL MANAGEMENT The temperature of the room and heat-retaining characteristics of the cages are important in studying old or CR animals, which have difficulty in adjusting to cold. Masoro (1991) discusses environmental conditions for aging rats, including the desirability of providing a room temperature some- what higher than normal. Given the limited knowledge in this regard, a room temperature of 25-27°C (77.0-80.6°F) is suggested for individually housed aging mice and rats, and a somewhat lower temperature for group- housed animals. Variables that will affect this recommendation are the characteristics of the caging (e.g., dispersion of heat through plastic versus through metal and the number of surfaces open to the air) and the airflow and air currents in the room (see Chapter 5~. As discussed previously, diet is a major consideration for aging ani- mals. It affects longevity, perhaps by influencing metabolism and certainly by influencing pathology. Not only caloric restriction, but also the effect of quantity and quality of the protein fed is important (Iwasaki et al., 1988), particularly for strains susceptible to kidney disease. One good high-qual- ity diet is NIH-31, which is used by NCTR for the NIA colonies and by institutions that use animals from the NIA colonies. Record-Keeping Record-keeping is discussed in Chapters 4 and 5. Some special consid- erations apply in aging rodent colonies. In long-term breeding colonies, records of paired-mated sublines should be kept so that selection for life- table characteristics can be either enhanced or limited. Careful records are obviously required for four- or eight-way matings and for the development of recombinant inbred strains. A few animals should be euthanatized and necropsied at regular intervals throughout the study. In the case of mice and rats, this process should begin no later than the age of 18 months. Transportation and Stabilization Aged mice and rats are especially susceptible to physical stresses, and this should be a consideration in shipping, as well as in housing the ani- mals. If animals are shipped in very hot or very cold weather, especially if there will be an intermediate holding period in an airport building, they can become debilitated or die. CR mice, in particular, have reduced resistance to cold because of their limited metabolic reserves. It is also difficult to maintain a diet regimen if shipping requires more than 24 hours. The best course of action is to pick up the animals at the airport as soon as they arrive. Transport cartons designed to protect against temperature changes and to maintain SPF status should be used. Arriving shipments of aged SPF rodents should be placed in a barrier facility immediately, even if they will be euthanatized soon after arrival. Failure to do so might lead to bacterial
RODENTS THAT REQUIRE SPECIAL CONSIDERATION 137 or viral infections that will affect physical performance, immune function, enzyme concentrations, standard blood values, or other characteristics that will be measured. A 2-week quarantine period should be imposed on all arriving shipments of aged animals before they are used in experiments to allow time for incipient infections, if present, to be expressed. Small (1986) has reviewed quarantine periods, particularly with regard to the introduction of communicable diseases (see also Chapter 6~. The value of a period to stabilize physiologic and behavioral responses probably varies with the study and should be established by each investigator. Veterinary Care and Surveillance Because there is an age-related decline in immune response (Miller, 1991), old mice and rats are especially susceptible to infectious diseases. Therefore, regular microbiologic monitoring (see Chapter 6) is essential for maintaining their SPF status. Sentinel animals should be used for monitor- ing because aged animals are usually too valuable to euthanatize or to sub- ject to multiple blood-collection procedures. Infectious agents of particular concern to gerontologists are mouse hepatitus virus, Sendai virus, rotavirus, and Mycoplasma pulmonis in mice and Sendai virus, Kilham rat virus, rat corona/sialodacryoadenitis virus, and Mycoplasma pulmonis in rats (Lindsey, 1986; NRC, 19911. Those agents are of concern because they affect either immune function or general health. Care of the animals and maintenance of their microbiologic status are usually overseen by the veterinary staff. However, to provide an early warning of incipient health problems, the research staff should observe each animal daily, including weekends and holidays. Moribund or dead animals should be picked up daily before postmortem changes make useful necropsy impossible. A full discussion of barrier facilities and surveillance programs and a summary of infectious disease agents and the systems that they affect have been published (NRC, 1991~. Important considerations to investigators who use aging animals are the timing and method of euthanasia of moribund animals. It is generally con- sidered inhumane to allow old and sick animals to die naturally; however, gerontologic research often requires an accurate record of the time of death. Even if a recorded time of death accurate only to within 24-48 hours would satisfy the experimental protocol, it is difficult to obtain because fragile old mice or rats can appear moribund for days or weeks before they die. Signs of imminent death that can be used to decide when to perform euthanasia are cessation of eating for 48 hours, reduction of body temperature (deter- mined by touching the animals with alcohol-washed fingers or measuring with an electronic thermometer), or maintenance of an immobile posture even if given a gentle stimulus. Each investigator should develop his or her
138 RODENTS: LABORATORYANIMAL MANAGEMENT own system with the guidance of the attending veterinarian and, having chosen it, should adhere to it rigorously. An advantage for the investigator of euthanatizing the animal is the ability to obtain usable tissue specimens and necropsy findings. Methods of euthanasia are discussed in Chapter 6. Other Rodent Species Used for Gerontological Research Other Species of Mus A number of interesting species of wild Mus and wild subspecies of Mus musculus are being adapted for laboratory use (Bonhomme and Guenet, 1989; Potter et al., 1986), but little is known about their life-table character- istics. Mus carol) (a rice-field mouse of Southeast Asia) is the single exception. Data on survival, reproductive life span, and age-related pathol- ogy have recently been published (Zitnik et al., 19921. The MxLS observed from among cohorts of 249 males and 231 females were 1,560 and 1,568 days, respectively. Gompertz analysis indicated an aging rate only slightly less than that published for wild Mus musculus. The shape of the survival curve (especially for females), however, suggests that many animals have died from causes not related to aging, such as fighting and acute stress. Peromyscus spp. The best studied member of the genus Peromyscus is Peromyscus leucopus, the white-footed mouse (Sacher and Hart, 1978), which has a life span about twice that of the laboratory mouse (Sacher, 1977~. Peromyscus, how- ever, is only "mouse-like"; it has been separated from Mus musculus for 15- 37 million years. Given that caveat, Peromyscus will continue to be useful in broader comparative gerontologic studies because it has adapted well to laboratory conditions. As with all such "domesticated" wild strains, how- ever, a substantial degree of genetic diversity is lost because of the small numbers of animals used to initiate laboratory populations. Guinea Pigs The guinea pig (Cavia porcellus) has been somewhat neglected by geron- tologists because of its comparatively large size, relatively long life span, and relatively high cost of maintenance. Although published survival curves have indicated an MxLS of around 80 months (Rust et al., 1966), some have recorded an MxLS of close to 10 years (Kunst'yr and Naumann, 19841. As with all iteroparous species (species that reproduce more than once in a lifetime) that have not been extensively used for research on aging, the MxLS is likely to be underestimated because record longevities are a func
RODENTS THAT REQUIRE SPECIALCONSIDERATION 139 lion of population size. At least three aspects of guinea pig biology make them of special interest to gerontologists: Like humans, guinea pigs are unable to synthesize ascorbic acid and so are candidates for studies of the free-radical theory of aging (Herman, 1986~; their cells appear to be resis- tant to transformation in vitro (like those of humans and unlike those of mice and rats) (T. H. Norwood and E. M. Bryant, Department of Pathology, University of Washington, Seattle, Washington, unpublished); and the con- siderable body of research that has been carried out on their auditory system (McCormack and Nutall, 1976) might provide useful background in studies on the pathogenesis of presbycusis. Guinea pigs are highly susceptible to a variety of infectious diseases; therefore, it is important to maintain them under SPF conditions for gerontologic research. Several such colonies have been established. Husbandry and dietary requirements of guinea pigs have been discussed in Chapter 5. Hamsters Primary cultures of Syrian hamster (Mesocricetus auratus) somatic cells are often used to study the cellular basis of aging. Cellular function, par- ticularly replicative capacity, can be analyzed in culture with a degree of experimental control that cannot be achieved in living organisms. Normal diploid somatic cells of all studied mammalian species initially divide rap- idly in culture, but the replicative capacity or life span of cells is limited, that is it eventually declines. Some of the cells from some species, how- ever, are spontaneously "transformed" and exhibit indefinite replicative po- tential. Transformation in primary cultures of mouse somatic cells is very rapid and difficult to study, whereas primary cultures of guinea pig somatic cells are resistant to transformation. Syrian hamsters exhibit transformation properties intermediate between those of mice and those of guinea pigs. Investigators interested in a manageable system for studying both the lim- ited replicative life span of cells and their ability escape from such a limita- tion have found this species to be useful (e.g., Bols et al., 1991; Deamond and Bruce, 1991; Sugawara et al., 19901. Recent data on survival and pathology are available for a colony of outbred male Syrian hamsters (Deamond et al., 19901. On the basis of 150 spontaneous deaths, the MnLS was 19.5 months, and the MxLS was 36 months. More than 35 inbred strains of Syrian hamsters have been de- scribed; most of these have not been carefully investigated in gerontologic research, and many are extinct. The Turkish hamster (Mesocricetus brandti), like other hamsters, offers an opportunity to investigate how hibernation might modify rates of aging and life span (Lyman et al., 19811. The direct correlation found between life span and the amount of time spent in hibernation is consistent with the hypothesis that one or more processes of aging are slowed during hiberna- tion (Lyman et al., 19811.
140 RODENTS: LABORATORYANIMAL MANAGEMENT Chinese hamsters (Cricetulus griseus) are of interest to cytogeneticists because their chromosomes are rather easy to study (Brooks et al., 1973~. Several outbred, inbred, and mutant stocks have been developed, but they are not as readily available as some other rodents. The life span character- istics of this species have not been rigorously investigated; however, al- though typical survival curves have been demonstrated for females, the curves for males, which usually live longer, are atypical. An MxLS of about 45-50 months has been reported for males (Benjamin and Brooks, 19771. Information on pathology is available for the colony maintained at the Lovelace Foundation Inhalation Toxicology Research Institute, Albu- querque, New Mexico (Benjamin and Brooks, 1977~. Husbandry and di- etary requirements have been discussed in Chapter 5. Gerbils Cheal (1986) has provided a comprehensive review of the Mongolian gerbil (Meriones unguicultatus) as a model for research on aging and has concluded that its ease of handling, ready availability, and particular physi- ologic and behavioral attributes establish it as a valuable model system. However, the gerbil exhibits an atypical survival curve (Figure 8.3), and much more must be learned about the causes for this, including susceptibil- ity to various infectious diseases and nutritional requirements. All gerbils in the United States are descended from only nine animals (Cheat, 1986), and there is some concern that deleterious recessive or dominant mutations might have become fixed in the population (M. Cheat, University of Dayton Research Institute, Higley, Arizona, unpublished). The husbandry of ger- bils is discussed in Chapter 5. RODENT MODELS OF INSULIN-DEPENDENT DIABETES MELLITUS With rare exceptions, the rat and mouse models of human autoimmune diabetes mellitus have appeared spontaneously, presumably as a result of mutation, rather than deliberate genetic manipulation. The discussion be- low focuses on two models of insulin-dependent diabetes mellitus: the BB rat and the NOD mouse. The management principles suggested are easily superimposed on standard rodent-management techniques. Diabetes-Prone and Diabetes-Resistant Rats In 1974, some animals were found in a closed colony of outbred WI rats (Big-Breeding Labs, Ottawa, Ontario) that spontaneously developed autoimmune diabetes mellitus (Chappel and Chappel, 19833. Several inbred diabetes-prone and diabetes-resistant strains were developed from this out
RODENTS THAT REQUIRE SPECIAL CONSIDERATION g ~ 50- FIGURE 8.3 Cheal (1986). 141 | Male Gerbil | - - - - - Male C57BU6J Mouse ~: - - - O- . 0 6 12 18 24 30 36 1 1 1 1 Months 42 48 Survival of conventionally reared male Mongolian gerbils. From bred stock at the Department of Pathology, University of Massachusetts Medical School. The diabetes-prone strains are designated BBBA/Wor, BBDP/Wor, BBBE/Wor, BBNB/Wor, and BBPA/Wor; the diabetes-resis- tant strains are designated BBDR/Wor and BB~B/Wor.1 The genetics and pathophysiology of the diabetes-prone strains have been reviewed (Guberski, 1993; NRC, 1989~. Breeding Techniques and Genetic Records Foundation colonies of diabetes-prone and -resistant strains are main- tained strictly by full-sib matings. However, the selection of litters from which future generations of breeders will be derived is influenced by the presence of desired phenotypic traits (e.g., incidence of diabetes, age at onset of diabetes, fertility, litter size, and survival of pups to weaning). Although it is recognized that the imposition of selection criteria can delay achieving inbred status, the goals of any breeding strategy must include preservation of the desired phenotypic characteristics (e.g., the development of diabetes mellitus). 1The designation BB/Wor was originally used as a group name for all seven inbred strains.
142 RODENTS: LABORATORY ANIMAL MANAGEMENT Essential data on each litter produced in the foundation colonies must be recorded to permit genetic tracing of breeding stock from one generation to another. To achieve this, a system of identification of each member of the primary and secondary breeding branches must be established. The records should include the occurrence of phenotypic characteristics, such as diabetes, thyroiditis, and lymphopenia. Husbandry and Care It is desirable that diabetes-prone and -resistant rats be maintained free of rodent pathogens in appropriate barrier facilities (see Chapter 5) because of the effect of these pathogens on phenotypic expression of diabetes (re- viewed by Guberski, 1993~. Microbiologic status should be monitored and recorded; records should include the tests performed and the frequency of testing. Experience has shown that these animals do well on a conventional light:dark ratio of 12: 1 2 hours. Detection and treatment of diabetes mellitus. The most cost-effective method of screening for diabetes is to test for glycosuria. Urine is expressed from the bladder manually by gently compressing the bladder against the pubic symphysis. Urinary glucose concentration is measured with a glucose test strip. Positive urine tests are confirmed with blood glucose measurements. Blood samples should be obtained from the tail within 2 hours of the urine test and tested with an appropriate technique. Animals testing 4+ for glycosuria and having blood glucose concentrations greater than 250 mg/dL are con- sidered diabetic. The age at which to begin testing and the frequency of testing for diabetes depend on the unique characteristics of the particular model and the environmental conditions under which it is kept. Testing for glycosuria should be started before the expected onset of diabetes and performed at least three times per week at the start of the light period in the light-dark cycles. The frequency of glycosuria testing can be reduced after about 120 days because new occurrences are less likely. Daily treatment of diabetic rats with insulin is mandatory and should begin on the day that glycosuria is found and diabetes is confirmed. The daily dose of insulin will be a function of age, body weight, the presence of ketoacidosis and dehydration, and the presence of pregnancy or lactation. Table 8.2 provides guidelines for the initial doses of insulin for animals that become diabetic after the age of 65 days. Animals that become diabetic on or before the age of 65 days should receive 0.2 U of insulin per 100 g of body weight in addition to the dose indicated. As animals increase in weight, the dose of insulin is increased by 0.2 U/10 g of body weight if the animals became diabetic on or before the age of 65 days, and by 0.2 U/16 g
RODENTS THAT REQUIRE SPECIAL CONSIDERATION 143 of body weight if the animals became diabetic after the age of 65 days. The maximal daily dose should not exceed 1.4 U/100 g of body weight for animals that became diabetic on or before 65 days of age, and 1.25 U/100 g of body weight for animals that became diabetic after the age of 65 days. If ketonuria (as detected with a test strip) develops, the dose of insulin should be increased, and lactated Ringer's solution with sodium bicarbonate should be administered in the amounts shown in Table 8.3. Injections of fluids are well tolerated when given under the loose skin on the back (distal to the nape of the neck). Treatment of hypoglycemia. Hypoglycemia is defined as severe if blood glucose is less than 40 mg/dL, moderate if blood glucose is 40-60 mg/dL, and mild if blood glucose is 60-80 mg/dL. The successful treatment of hypoglycemia requires a decrease in insulin dose combined with subcutane- ous injections of fluid. Suggested regimens are outlined in Table 8.3. Care of pregnant females. If ore anant animals become aglycosuric, the ~.. . . ~ _ course of action depends on the ratio of insulin to "ideal" body weight TABLE 8.2 Starting Doses of Insulin for BB/Wor Rats That Become Diabetic After the Age of 65 Days Initial Blood Glucose Concentration, m: /dL 250 300 350 400 450 500+ Body weight, ga Starting Dose of Insulin,b U 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 0.4 0.4 0.6 0.8 1.0 1.2 1.4 1.4 1.4 1.6 1.6 1.8 2.0 _. _ 2.2 0.6 0.6 0.8 1.0 1 ~ . _ 1.4 1.6 1.6 1.6 1.8 1.8 2.0 2.2 2.4 2.4 0.6 0.6 0.8 1.0 1.2 1.4 1.6 1.8 1.8 2.0 2.0 2.2 ~ 4 _. 2.6 2.6 0.6 0.8 1.0 1.2 1.4 1.6 1.8 1.8 2.0 2.0 2.2 2.2 2.4 2.6 2.8 0.8 0.8 1.0 1.2 1.6 1.6 1.8 2.0 2.0 _. _ 2.2 4 _. 2.6 2.8 3.0 0.8 0.8 1.2 1.4 1.6 1.8 2.0 2.0 2.2 2.2 2.4 2.4 2.6 3.0 3.2 aAssumes that rat is well hydrated and that ketosis? if present. is being corrected. bPZI U40 (Eli Lilly) insulin and a U/100 Lo-dose syringe (B-D) are used. U40 insulin + U./ 100 syringe = 0.4 units per gradation mark. Add 0.2 U/lOO g of body weight to the dose for animals that develop diabetes on or before the age 65 days. Maximal daily dose equals 1.4 U./ lOO g of body weight for animals that become diabetic on or before the age of 65 days and 1.25U/100 g of body weight for animals that become diabetic after the age of 65 days.
144 TABLE 8.3 Treatment for Ketonuria in Bl3/Wor Rats RODENTS: LABORATORYANIMAL MANAGEMENT Increased Insulin,a Lactated Ringer's Sodium Bicarbonate, Ketones U/100 g body wt Solution' cm3 mEqb 2+ 0.2 10.0 0.0 3+ 0.2 9.0 1.0 4+ 0.2 1 8.0 2.0 aInsulin dose of lactating females should not exceed 1.0 U/100 g of "ideal" body weight (see Care of pregnant females). Dose should not be increased during mild episodes of ketonuria. bl cm3 of 8.4% sodium bicarbonate equals 1 mEq. SOURCE: Guberski, 1993. (IBW). The IBW of a pregnant female at the age of 90 days is considered to be 270 g. If the animal is more than 90 days old, the body weight of a nonpregnant female sibling should be used as the IBW. The following procedures are recommended: . If the ratio of insulin to IBW is greater than 1.0 U/100 g, the dose of insulin should be reduced by 15 percent. . If the ratio of insulin to IBW is 0.9-1.0 U/100 g, the dose of insulin should be reduced by 10 percent and 10 cm3 of lactated Ringer's solution should be administered. · If the ratio of insulin to IBW ratio is less than 0.9 U/100 g, the dose of insulin should be reduced by 0.2 U/100 g and 10 cm3 lactated Ringers solution should be administered. If pregnant animals are severely hypoglycemic, follow the instructions for treating hypoglycemia in Table 8.4. If a female becomes ketotic at parturition, the insulin dose should not be changed. Instead, lactated Ringer's solution and sodium bicarbonate should be injected subcutaneously in the amounts indicated in Table 8.3. Care of lactating females. Beginning 12-14 days after delivery, insulin should be decreased by 10-15 percent each day until a dose of 0.8-1.0 U./ 100 g of IBW is achieved. To prevent hypoglycemia in lactating females, food should be made readily accessible by placing it on the cage floors. If hypoglycemia occurs, it should be treated as indicated in Table 8.4. Use of Spleen Cells to Reduce Frequency of Diabetes and Improve Breeding Efficiency Diabetes-prone rat strains are profoundly T-cell lymphopenic. Injec- tions of neonatal bone marrow, fresh spleen cells, or concanavalin-A-stimu
RODENTS THAT REQUIRE SPECIAL CONSIDERATION TABLE 8.4 Treatment for Hypoglycemia in Diabetic BB/Wor Rats Classification (blood glucose concentration) Subcutaneous Fluid Therapy Severe Give 1 cm3 50% dextrose; (<40 mg/dL) 2 hrs later give lactated Ringer's solution with 5% dextrose Moderate (40-60 mg/dL) Mild (60-80 mg/dL) SOURCE: Guberski? 1993. Give 10 cm3 lactated Ringer's solution with 5% dextrose Give 10 cm3 lactated Ringer's solution 145 Change in Insulin Dose Reduce by 30-50% Reduce by 20-30% Change in Time of Insulin Administration Delay by 2-3 hrs Delay by 2-3 hrs Reduce by 10-15% No delay lated spleen cells correct the T-cell lymphopenia and substantially reduce the frequency of spontaneous diabetes (Naji et al., 1981; Rossini et al., 1984~. Fresh spleen cells are obtained from diabetes-resistant rats, which are histocompatible with diabetes-prone rats but are not lymphopenic. Spleens are prepared with standard techniques (Burstein et al., 1989~. Diabetes prone rats between 21 and 40 days old receive one spleen equivalent of fresh donor cells in 1 cm3 of RPMI medium 1640, administered intraperito- neally. This procedure reduces the incidence of diabetes from greater than 85 percent to about 15 percent. Nondiabetic females do not require daily insulin injections (this reduces the workload of the staff) and are more productive breeders, as shown in Table 8.5. Shipping Pathogen-Free Rats Diabetes-prone rats have severely compromised immune systems and should be shipped in crates designed to keep them free of rodent pathogens (see Chapter 61. Drinking water or a water-rich material must be provided, especially for diabetic rats showing signs of polydipsia and polyuria, be- cause these animals are prone to dehydration. Commercial carriers should be instructed to use climate-controlled trucks and holding rooms because diabetic rats are more susceptible than normal rats to fluctuations in tem- perature. In addition, commercial carriers must guarantee delivery within 24 hours because shipping delays are hazardous for animals that require daily insulin injections.
146 RODENTS: LABORATORYANIMAL MANAGEMENT TABLE 8.5 Reproduction in Diabetes-Prone BB/Wor Rats Before and After Receiving Splenocytes from Diabetes-Resistant BB/Wor Rats Diabetes-Prone Females Not Treated with Splenocytes (N = 1,238) Diabetes-Prone Females Treated with Splenocytes (N = 1,022) Incidence of diabetes 86% 16% No. pups born 7,160 12,434 No. pups weaned 5,766 10,918 Pup survival through weaning 80.5% 87.8% No. pups weaned per female mated 4.7 10.7 SOURCE: Guberski, 1993. NOD Mice NOD (nonobese diabetic) is an inbred strain derived from Jcl:ICR mice with selection for the spontaneous development of insulin-dependent diabe- tes (Making et al., 1980~. The expression of diabetes in this strain is under polygenic control (Letter, 19931. Clinical features of diabetes in NOD mice are similar to those in humans. Females develop diabetes at a higher inci- dence and at an earlier age than males. The genetics and pathophysiology of this model have been reviewed (Letter, 1993; NRC, 1989~. Insulin treatment is required to maintain diabetic NOD mice; without insulin, they survive only 1-2 months after diagnosis. Diabetes is diag- nosed by determining that the blood (nonfasting) or plasma glucose concen- tration is increased. This determination can be made by measuring blood glucose directly or by measuring urinary glucose with a glucose test strip. Glycosuria, as read on the test strip, usually denotes a plasma glucose of 300 mg/dL. Large numbers of mice can be easily screened by this method. It is difficult to keep serum glucose within a normal range with insulin treatment, but body weight can be maintained and life prolonged (Ohneda et al., 1984~. Morning and evening intraperitoneal injections of a 1:1 mixture of regular and NPH insulin are satisfactory. The dose will be 1-3 U. de- pending on the extent of glycosuria. Environmental factors are extremely important in the expression of diabe- tes in NOD mice. Keeping them in an SPF environment increases the occur- rence of diabetes; exposure to a variety of murine viruses, including mouse hepatitis virus (Wilberz et al., 1991) and lymphocytic choriomeningitis virus (Oldstone, 1988), prevents diabetes development. That various types of exog- enous immunomodulators prevent the development of diabetes (Letter, 1990J suggests that infectious agents prevent diabetes by general immunostimulation. Diet also has an important effect on diabetes development: natural-ingredient
RODENTS THAT REQUIRE SPECIAL CONSIDERATION 147 diets, including standard, commercially available mouse feed, promote a high incidence of diabetes (Coleman et al., 1990~. NOD is an inbred strain and should be maintained by brother x sister mating. NOD mice have an excitable disposition but breed well. Siblings bred before the development of overt diabetes can usually produce two large litters (9-14 pups each) of which nearly all the pups survive to wean- ing. Breeders can be protected from developing diabetes by a single injec- tion of complete Freund's adjuvant (Sadelain et al., 1990~. TRANSGENIC MICE Since the late 1970s, advances in molecular biology and embryology have enabled scientists to introduce new genetic material experimentally in-to the germ lines of mice and other animals. The term transgenic mice, as used here, means that foreign DNA has been introduced into mice and is transmitted through the germ line. The gene transfer can be performed to introduce new genetic traits or to negate or "knock out" host-gene function by targeted mutagenesis. Foreign genetic sequences can be introduced into mouse cells, espe- cially in early embryos, by several different methods. The most commonly used method is pronuclear microinjection, in which a solution of purified DNA is injected into either of the two pronuclei visible in a newly fertilized egg (Gordon et al., 19801. Other, less reliable methods include the carrying of the proviral DNA into the cell with a retroviral vector (Jaenisch, 1976) or by electroporation (Toneguzzo et al., 1986) and transformation of toticotent embryonic stem (he) cells, which are derived from cultured blastocyst- stage embryos (Doetschman et al., 1987~. In contrast with microinjection or retroviral insertion, integration of foreign DNA into ES-cell chromo- somes can be targeted to specific loci. The specifically modified, undiffer- entint~.~1 F.S c.~11s can then be introduced into a recipient embryo in which (it ~ ~ ~ This ap- proach is used not only for modifying gene expression, but often for intro- ducing targeted mutations by replacement of genes with nonfunctional coun- terparts, that is, for producing "knockouts" (Mansour et al., 1988~. _ ^, ~, ~ _ _ ~ _ ~ . is hoped) they will incorporate into the developing germ line. Colony Management Although a transgene causes only a small change in a genome, it can produce dramatic and unpredictable changes that make colony maintenance a challenge. Husbandry and production of transgenic mice have been re- viewed (Gordon, 1993) and will be described briefly here. Colony management can be complicated by several characteristics of transgenic mice, including unpredictable phenotypic effects of transgene expression, pathologic effects of the transgene that compromise viability,
148 RODENTS: LABORATORYANIMAL MANAGEMENT unpredictable interactions between the transgene and other host genes (e.g., insertional mutagenesis), altered responses to microorganisms or other envi- ronmental variables, compromised fertility, and possible instability of transgene expression through generations. Depending on the presence and severity of those characteristics, barrier maintenance might be advisable. Filter-top caging systems are usually sufficient if proper precautions are taken. Flex- ible-film or rigid isolator systems, however, permit the most complete con- trol of the physical and microbiologic environment. Microbiologic status should be monitored regularly and should include testing for standard mu- rine infectious agents. Both transgenic and sentinel mice should be evalu- ated if the integration or expression of a foreign gene alters immune compe- tence. Transgenic mice should be observed daily, and all visible clinical events should be recorded. Animal-care technicians should be trained to recognize clinical events and to report their occurrences with appropriate descriptive terminology. Unexpected deaths should be discussed with an animal-health professional, such as an animal pathologist, to determine whether necropsy and histologic examination are warranted. It is imperative that deceased animals be collected and preserved properly as soon as they are discovered. Corpses can be placed in fixative, refrigerated, or frozen, depending on the specific postmortem procedures that are planned. Management of a transgenic-mouse facility includes special require- ments for embryo donors, embryo recipients, and offspring. In many transgenic facilities, embryo collection and culture, DNA introduction, and embryo transfer are performed outside the barrier; therefore, the embryos and em- bryo-transfer recipients might no longer be SPF and should not be returned to the barrier. Embryo Donors Embryos into which DNA will be introduced to generate founder mice are obtained by administering exogenous gonadotropin hormones intraperi- toneally to virgin females. The hormones elicit synchronized ovulation of a relatively large cohort of mature oocytes (i.e., superovulation); therefore, fertilization and later preimplantation development will also be synchro- nized. Very young females 28-40 days old, depending on the stock or strain usually respond best to superovulatory hormones. Outbred mice were originally used as embryo donors; more recently, inbred FVB mice have also been used. FVB mice are highly inbred, they respond well to superovulatory hormones, and their embryos have large pronuclei (Taketo et al., 1991). Males should be individually housed; females can be group-housed be- fore mating. Breeding is most effective if a 3- to 8-month-old male that is a
RODENTS THAT REQ UIRE SPECIAL CONSIDERATION 149 proven breeder is paired and bred with one or two females every 2 or 3 days. Mating should always occur in the cage of the male. An uninterrupted dark phase of the lighting cycle is critical for efficient superovulatory breeding; a light:dark ratio of 14 to 10 hours is effective. Two gonadotropin hormones, pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (HCG), are each administered 7-9 hours before the beginning of the dark cycle, but PMSG is administered 2 days before HCG. Pronuclear embryos are generally collected 14-17 hours after the beginning of the dark cycle. For example, if the dark cycle begins at 10 p.m., PMSG would be administered between 1 and 3 p.m. 2 days before the day of mating, HOG would be administered between 1 and 3 p.m. on the day of mating, and pronuclear embryos would be collected between noon and 3 p.m. the next day. Embryo Recipients Group-housed females are used; outbred or hybrid mice generally make the best dams. Good choices of stocks to carry transferred embryos include outbred ICR mice (if a white coat is desiredJ and C57BL/6 x DBA/2 F1 (B6D2F1) hybrid mice (if a colored coat is desired). Housing strategies that avoid synchronization of estrus in group-housed females have been described (Gordon, 1993~. A colony of vasectomized males is required. It is preferable for the males to be test mated to ensure sterility; however, if 5- to 6-week-old males are vasectomized, there is no sperm yet in the vas deferens, and test mating is not necessary. Even if test mated, males used to produce pseudopregnant females should be a different color from the embryo donor so that "acciden- tal" offspring of males that have recovered their fertility can be distin- guished from transgenic offspring. Embryo-donor females should be 0-1 day more advanced in the repro- ductive cycle than pseudopregnant females. Early (one or two cells) em- bryos are transferred into the oviduct of the embryo recipient; morula and blastocyst embryos are transferred directly into the uterus. Recipient fe- males should be used only once. Ofi~spring Individual litters should be separated by sex at weaning and housed in cages that clearly indicate the litter number, date of birth, lineage, and parental identities. In general, fewer than 25 percent of live-born pups that receive transgene DNA as embryos will have integrated transgenes; 10 per- cent is considered average if microinjection is used. Most transgenic mice are identified by Southern blotting or polymerase chain reaction (PCR) analysis
150 RODENTS: LABORATORYANIMAL MANAGEMENT of DNA extracted from tissue taken from the tip of the tail; approximately 1 cm of tissue is sufficient. Rarely, it is possible to identify transgenic mice by detecting gene products from the introduced DNA. Breeding Transgenic Mice Once a mouse is identified as transgenic, it should be bred to verify that the transgene has been integrated into its germ cells. The development of a colony of mice homozygous for the transgene is achieved by standard breeding and test-mating procedures. Homozygous transgenic mice will produce 100 percent transgenic progeny on mating with a nontransgenic mate, whereas hemizygotes will produce both transgenic and nontransgenic offspring. It is recommended that multiple test litters be analyzed before the homozygosity of a breeder is considered established. Transgenic inheritance patterns do not always conform to classical Mendelian patterns, because the integration and expression of a transgene can affect implantation, in utero develop- ment, and postnatal survival. When mice are not homozygous for the transgene, all offspring must be screened for the transgene. Reproductive performance of transgenic mice can differ substantially from that of the nontransgenic parental or background strains. Insertional phenomena can compromise fertility and affect embryo survival. Although breeding mice to homozygosity for the transgene is often desirable, ho- mozygotes might be inviable, infertile, or subfertile. If fertility problems are encountered in homozygotes, whether caused by transgene expression or insertional mutagenesis, the problem can often be effectively managed by maintaining the transgene in the hemizygous state. Even in hemizygous mice, however, the effects of transgene integration, transgene expression, or both can be detrimental to survival and reproduction, and investigators and animal-care personnel should be alert to the necessity for establishing ag- gressive breeding programs. In extreme cases, assisted-reproduction tech- nologies (e.g., superovulation and in vitro fertilization) might be helpful. Identification, Records, and Genetic Monitoring Identity, breeding, and pedigree records must be fastidiously kept be- cause breeding errors in transgenic colonies are difficult to detect. For example, classic genetic monitoring will not necessarily distinguish between different transgenic lines on the same background strain. Even direct ex- amination of the transgenic DNA sequence (e.g., with Southern blotting or PCR analysis) might not definitively identify a specific mouse. It is recom- mended that a combination of methods for identification and genetic moni- toring be used in a colony of transgenic mice. Purified DNA samples from important animals can be frozen and stored at -70°C; these might be useful for future analyses, especially if DNA rearrangement is suspected.
R ODENTS THA T REQ UIRE SPECIAL CONSIDERA TI ON 151 Individual animals can be marked rapidly and inexpensively by tattooing, clipping ears, or using ear tags. The most reliable, albeit most expensive, system for identifying an individual animal is subcutaneous implantation of a transponder encoded with data on the animal. Transponder identification chips are durable for the life of the animal and suitable for computerized data- handling. Whatever method is chosen should be used in conjunction with a well-maintained cage-card system. One issue that arises in colonies of geneti- cally engineered animals that does not arise in other colonies is confidentiality specifically related to patentability of the animals; information displayed on cage cards should be reviewed with the principal investigator. The identity of each transgene-bearing breeder should be verified be- fore mating Imnortant information on the transgenic parent includes tran ~ vat ~ '''~''^~4t~ ~ ~ ~-~~ ~ ~~^ ~ ~ - ^ ~ ^~~ ~~ ~ ~~ ~ ~~ ~~~ ~ ~- ~~--- (~ - ~ spender code or other identification code, lineage, date of birth, date of pairing, administration of exogenous hormones (if any), and date of separa- tion of breeding pair. If mice escape, all unidentifiable animals should be euthanatized, and recaptured identifiable females should be isolated for at least 3 weeks to determine whether they are pregnant. Litters derived from questionable or unverified matings should be euthanatized. Embryo Cryopreservation Because each transgenic line is unique, embryo cryopreservation might be considered. In general, cryopreservation issues relevant to transgenic lines are the same as those relevant to other rodents (see Chapter 41. How- ever, some lines cannot be made homozygous, are reproductively compro- mised, or both, so it might be prudent to freeze more embryos than would be necessary for preservation of an inbred strain. Data Management A large amount of data accumulates in a transgenic colony and must be managed efficiently. ~ ~ ~ Daily or weekly records Include data on oreeolng birth, weaning, death, and laboratory analyses; they also include documen- tation of observations on such things as characteristics that are possibly related to gene manipulation, pathologic conditions, and unusual behaviors. Shipment and Receipt of Transgenic Rodents In general, it is not necessary to use extraordinary containment proce- dures for shinning trans~enic mice. '' ~ ~ To reduce the risk of loss, shipments can be split so that accidents or errors during transit do not compromise the entire shipment. The following information should accompany transgenic mice shipped from a facility and be requested for transgenic mice brought into a facility:
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