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a Department of Pathology and Geriatrics Center, University of Michigan School of Medicine, Institute of Gerontology, and Ann Arbor VA Medical Center, Michigan
b National Institute on Aging, Bethesda, Maryland
Richard A. Miller, The Geriatrics Center, University of Michigan, CCGCB, Room 5316, Ann Arbor, MI 48109-0940. E-mail:millerr@umich.edu
Decision Editor: Jay Roberts, PhD
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Abstract |
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THIS article is intended to convey some rules-of-thumb for investigators just starting to think about the design of experiments on aging using mice and rats. The principles stated below reflect the opinions of the authors, based on years of experience in rodent-based work in gerontology and molecular biology. However, to avoid excessive circumlocution of the "in my opinion it may be helpful to" variety, the style is deliberately imperative, modeled on examples set by writers of style manuals (1) and columns of advice for the lovelorn. In addition the word "mice" will be used throughout to mean "mice and rats," except in those cases where mice are different from rats.
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First Principle: Don't use mice that are too old. |
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(a) Old mice are usually sick (even if not quite dead yet). If a trait differs from young controls only in the last 10% of the cohort to drop off, then it's hard to be confident that the change is a result of aging rather than of the advanced disease or diseases most typical in the stock under study. After all, the aging process, which creates decrepit old mice from healthy, fit, young ones, takes many months to do this, and age-dependent changes in many cells, tissue, and organ systems can usually be demonstrated well before the median survival time for the species or stock. If your assay shows no change at 18, 22, 26, or 30 months of age (in a stock with a median survival of 24 months), then demonstrating a change in 34-month-old animals may well be due to sickness per se. Judicious selection of ages for initial exploratory work may depend on the specific characteristics of the stock to be used, and stocks with median survivals of 22 months or of 30 months may call for adjustments of ages selected for initial examination.
(b) Old mice are very expensive, particularly if you want them to be disease-free. The problem is that the real production cost of mice rises not linearly with chronologic age, but instead in proportion to the mortality rate, i.e., as an exponential function of age. If half your mice live to age 24 months, then producing a single 24-month-old mouse requires you to pay someone to house two mice for 24 months, one of which has just died. If only 10% of the mice survive to age 32 months, then the real cost of each 32-month-old mouse is the cost of raising 10 mice for anywhere from 18 to 32 months to get the one alive at 32 months. And then that one mouse, when you do the necropsy, may well turn out to have advanced neoplasia.
To illustrate the projected costs, at one well-known Midwestern Medical Center, animal users are charged $0.58/cage/day for cages of four mice. At this price it costs $106 to grow a mouse for 2 years; but because half the mice die, the cost of a live 2-year-old mouse is twice as high, or about $212. Because half of the 2-year-old mice are found to have advanced neoplasia even at a cursory necropsy, the cost of a more-or-less tumor-free 2-year-old mouse is another twofold higher, or $414. The nominal cost of a 32-month-old mouse (at $0.58/cage/day) is $140, but adjusting for attrition and disease gives a real cost closer to $1,400 each.
The National Institute on Aging (NIA) Office of Biological Resources provides highly subsidized animals, even with the recent price increase; for a 2-year-old C57BL/6 mouse, for example, the cost charged to investigators is a mere $72, well under the local production cost. The real subsidy (production cost minus cost to you) rises exponentially with age, however, and therefore it costs much more to raise a very old mouse in one's own facility than it does to buy them from NIA. Although NIA continues to raise these very old mice, despite their exceptionally high cost, in order to provide investigators maximal flexibility in designing their experimental protocols, investigators who do use this scarce resource are still confronted with the problems imposed by their very high rates of concurrent illness.
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Second Principle: Don't use mice that are too young. |
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Third Principle: Don't use too few age groups. |
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Fourth Principle: The mice must be specific pathogen–free, and you have to be able to prove it. |
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These operating procedures, although necessary, are not sufficient to earn the right to refer to your colony as SPF; the colony must be proven to be free of key pathogens by routine testing of surveillance mice, for example on a quarterly basis. A minimal surveillance program involves introducing several cages of new mice, without filter bonnets, into each room every three months, using a stock (CD-1, for example) known to be susceptible to many common pathogens. These mice are then tested after 90 days, a period of time sufficient to allow them to become infected by anything in circulation, and to have developed antibodies to the infectious agent. The mice are then euthanized, examined carefully for evidence of intestinal and external parasites, and their sera tested (often by a commercial laboratory) for evidence of antiviral antibodies specific for the agents of interest. Periodic histopathological analysis is also recommended. If all tests come back negative, then you can call the colony SPF. Positive responses should induce a panicky feeling and a vigorous retesting effort, and repeat positives usually require that the entire affected colony be discarded and rederived.
If this surveillance system is not practicable in a given institutional vivarium, a useful alternative is to keep a small number of weanlings from breeding cage until they are about 3 months old and then to send these to a commercial facility for testing. Because the breeding pairs are usually long-term residents of the facility, and newborn pups are particularly sensitive to infection, this procedure is more sensitive than buying mice and allowing them simply to reside in the colony for a few months without contact with the local residents.
The optimal situation for maintaining an SPF colony combines the use of filter bonnets with the use of sentinel mice. Because filters work very well in preventing spread of airborne pathogens, exposure of the sentinel animals to potentially infectious agents requires a procedure in which the used bedding from a pool of cages be thoroughly mixed, and then added to the cages containing the sentinels. (A policemouse's life is not a happy one.) Sentinels who put up with this treatment for several months are then volunteered for necropsy and serological analysis. In these circumstances a stray positive result may not require sacrifice of the entire colony, because it is more likely that the infection has been confined to one or two cages; detailed follow-up studies may document good health for the majority of mice in the room. To be effective, this system requires good record-keeping in order to trace all cages that a sentinel has had contact with.
Why go through this hassle? "After all," the scientist stuck with a conventional colony might rationalize, "people are not free of all infectious agents; I'm just trying to more closely mimic the real world situation." The basic problem is that the intensity, variety, and prevalence of infection in any given conventional colony may well change from month to month and year to year, and is likely to differ greatly from one colony to another. Because many infections can alter a mouse's immune, hepatic, endocrine, digestive, pulmonary, and neurological responses, studies carried out on conventional colonies can prove very difficult to reproduce in another, or even in the same, laboratory. In some cases allegations of age effects on variables of interest have proven to occur only in conventional colonies (3), and are thus likely to reflect unsuspected influences of one or more uncharacterized infectious agents than of aging itself. Most effects of this kind doubtless go undetected, because few workers routinely use mice from two distinct colonies, one conventional and the other SPF. It seems likely, however, that many of the unnerving conflicts among reports in the gerontological literature may reflect variations in colony pathogen status.
Successful maintenance of an SPF colony also requires sufficient discipline to prevent the importation of new mouse stocks from uncertified suppliers. A well-run colony will usually permit unfettered importation of mice from only a very small number of commercial vendors, vendors that routinely submit clean bills of health with all shipments. A request for permission to bring in animals from an uncertified vendor or another research institution should trigger a process in which the sender is required to document the health status of the animals, and in which even allegedly clean animals are kept in a separate quarantine facility (or building) until tested locally for pathogens before they or their offspring are introduced into the general population. Importation of a stock that cannot be proven SPF ordinarily requires long-term quarantine or rederivation of the stock by caesarian delivery and foster nursing.
Two common mistakes: Scenario 1—the vivarium manager tells you it's an SPF colony, because the facility only buys from SPF suppliers. So why spend the money to test this? Four years ago, however, your technician visited a pet store on the way to work and every cage has had Sendai for four years—your laboratory mice are about as SPF as the ones in your basement. Test quarterly, and you can proudly report the clean bill of health in every paper and every grant proposal.
Scenario 2—you have a conventional colony, and you're not proud of it, but you buy the SPF mice from NIA and let them sit in the colony for just a week or two before use. This approach, which is remarkably common, just about guarantees that the mice used in your tests are infected with something; blessed with an SPF upbringing, they have no protective antibody titers, and are thus sitting ducks for whatever virus happens to be in your colony during their initiation into the tough realities of real world infection. Even if the mice will be housed in the animal facility short-term, it is well worthwhile to use an SPF facility or at least a quarantine room and filter bonnets.
It is not a good idea to try to sneak past the problem in Scenario 2 by using the mice the day they arrive off the delivery van. Shipping is very tough on mice, and the stress has an impact on adrenal size, steroid hormone levels, immunity, and other organ systems. No one would write a protocol that began, "Prior to their use in experiments, the mice were placed in a shipping container without access to their usual sources of food and water, and then flown 1000 miles in a dark, cold, noisy plane, followed by interstate truck shipment..." It's a good idea to let the mice sit after arrival, for at least a week or preferably two, before use. But do it in an SPF colony.
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Fifth Principle: Don't bet the farm on C57BL/6 mice. Don't bet it on F344 rats, either. |
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• All mice in an inbred stock are genetically identical. It's therefore impossible to be certain that conclusions based on an inbred stock will apply equally to any other inbred stock without doing the study all over again.
• Inbred stocks are not only homogeneous, they are also weird, debilitated, and short lived. Creation of an inbred stock involves forced homozygosity at all loci. This is a highly selective process, because the inbreeding process frequently creates genotypes that impair viability and fertility; in fact, most brother-sister–mated families eventually die out, with the few surviving families becoming the "standard" inbred lines we all know and love. F1 hybrids created by a cross between two different inbred lines are almost invariably longer-lived than either of the two parents (7), consistent with the notion that the homozygous condition produces an animal of lower quality. Many inbred lines are famed for properties that clearly count as strain-specific oddities: the chronic renal disease and high lymphoma incidence of the F344 rat, the 100% incidence of thymic lymphoma in the AKR/J mouse, the near 100% incidence of reticulum cell sarcoma in SJL/J mice, and many other similar peculiarities. Table 1 shows a series of anecdotes; the take-home message is that individual inbred lines may have a very high incidence of lesions that are rarely seen in other inbred lines. These obvious illnesses, and other idiosyncrasies less obvious to the naked eye, could in principle wreak havoc on the process by which general conclusions are inferred from a limited data set. In some cases, of course, genetic identity is critical to the experimental plan; these situations include protocols that involve transfer of tissues from one mouse to another, and those where the goal of the study involves analyses of interstrain variation. But in many other instances the use of an inbred strain reflects mere custom rather than a careful decision among alternatives. To read more along these lines, check out R. Weindruch's article (5).
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(a) If you have to use genetically homogeneous animals, prefer F1 hybrids to inbred mice. Although each individual F1 stock is genetically uniform, at least you've ducked the homozygosity problem, and F1 mice are in general longer-lived, hardier beasts.
(b) Replicate key findings in multiple stocks. Once you've invested three years in proving something in CB6F1 mice, it may seem a waste of time to spend another few months checking the main points in two other F1 lines, but in the long run this may be more productive than spending the rest of your career chasing a finding that turns out to apply only to CB6F1.
(c) Consider the use of an animal stock with controlled heterogeneity, such as mice bred by a four-way cross (e.g., CB6F1 mothers crossed to C3D2F1 fathers). Such a cross yields an arbitrarily large group of full sibs; no two mice are genetically identical, but each mouse shares half of its genetic code with any other randomly chosen animal in the pool. There is a slowly growing literature demonstrating the usefulness of such heterogeneous lines in aging research, and the NIA Office of Biological Resources plans to add such mice to their contract colonies soon.
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Sixth Principle: Do at least a quickie autopsy on each old mouse; if you can afford it, pay a pro to do a gross necropsy. |
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Having a look yourself is better than not looking, but better still is to give the animal, after you've taken the tissue you need, to a veterinary pathologist or technician and ask them to have a look. This is fairly inexpensive, you get the written report back in a few weeks, and then you can go back through your notes and discard the data that came from the ones later found to have a serious illness.
Best of all is a histopathological autopsy. A thorough job costs $50–$100 per case; a quick microscopic look at the obvious lesions can cut the cost to $25 or so. This is very important if you are characterizing a new model (does the drug you administer to prevent neurodegeneration increase the incidence of liver abnormalities?). But if your main goal is to eliminate data from sick mice, the gross inspection is often adequate and much cheaper.
An alternate, more informative, approach would be to consider whether the presence of a specific form of illness modifies the age effect or treatment effect under study. Limited statistical power—are the differences between tumor-ridden and tumor-free mice big enough to be worth separate analysis—and the difficulties of deciding the extent to which similar disease states can safely be lumped together can greatly complicate this variety of analysis. In any case it is important for the investigator to state explicitly the criteria used for elimination (or stratification) of individual animals, and the proportion of animals that met the inclusion criteria.
At what ages is the yield worth the cost? This will vary from strain to strain, and if you find that 95% of your 16-month-old mice are free of lesions that might compromise your interpretation, then you may want to skip paying for the gross inspections and do it yourself. As a rule of thumb, we try to get a gross necropsy on mice over 18 months and rats 24 months or older. Your mileage may vary.
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Seventh Principle: Don't pool unless you absolutely have to. |
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The second issue is a statistical one: the assessment of a statistical hypothesis ("mice aged 18 months express, on average, more of this gene than mice aged 6 months") depends on the number of individual mice, or pools of mice, tested independently. Thus an experiment in which a pool of 20 young mice is compared to a pool of 20 old mice has no greater statistical power than an experiment comparing one young to one old animal, i.e., none at all. The fewer mice used in each pool (ideally one mouse per pool), the more statistical power is achieved for the available mouse budget. Putting in the extra effort to miniaturize your test system to the point where it can be performed with material from a single mouse pays off handsomely in the long run.
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Eighth Principle: Buy extra old mice to compensate for death and disease. |
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Ninth Principle: Do a cost-adjusted power analysis and save a bundle. |
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, you've got only a 58% chance of getting a significant result if the real difference is 200 µg. To get an 80% chance of detecting a difference of 200 µg, you're going to need to include 17 mice in each group. You then go out and buy some extra mice. OK, next you want to know if the size of this muscle varies with age rather than with gender. So you do the same calculation, and get the same result: to get 80% power for detecting a difference between young and old of 200 µg, given the same assumptions above, you're going to need 17 young mice and 17 old mice. You go out and buy these 34 mice; actually you buy 17 young and more than 17 old, because some of the old animals will have to be discarded when you find out they have tumors.
Mistake: you've just wasted some money. The power analysis was done to calculate the minimum number of animals, but what you really want to do is get the maximal amount of statistical power per dollar spent. Because the old mice cost a lot more than the young ones, the cheapest way to get this statistical power is to buy slightly fewer old mice, and a good deal more young ones. If, for example, the cost of studying each young mouse (purchase cost plus cost of doing the assay) is $18, and the cost of studying each old mouse is $106, then the optimal solution is to buy 29 young mice and 12 old ones. If you buy 17 of each, you'll spend $2108; if you buy 29 plus 12, you spend $1797. You can allocate the $311 to the next experiment, or give your tech a well-earned raise.
Actually, the real savings can be very high indeed, particularly if you have to grow the mice yourself rather than obtain them from the NIA's highly subsidized colonies, and particularly if you count in the cost of the mice you couldn't use because they had serious disease. The real cost of a 2-year-old mouse is not $106, but the $212 you spent to grow two of them, the one that died last week and one you've still got, or often the $424 you need to spend to get a tumor-free mouse. At $424 per old mouse, the minimal cost is to study 50 young and 10 old mice, at a cost of $5242 and a savings of $2272. At the NIA subsidized cost, you'll pay a mere $72 a head for the old mice, or $144 per tumor-free old mouse, and $22 per young one; so you'll save a mere $454—less cost, and less savings than the home-grown variety, but still worth the cost of writing down the following formulas, which were derived by Andrzej Galecki of the University of Michigan's Geriatrics Center and Institute of Gerontology:
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Here's what to do: first do the regular old power analysis, that tells you how many mice you'll need if you use equal numbers of mice in each group. This was 
mice per group in the example shown above. Then figure out how much it costs to do the assay for each young mouse, including purchase costs, supply costs, tech time, overhead; this number is CY, the cost per young mouse. Calculate CO, the cost per old mouse, in the same way, and be sure to throw in the adjustment for the number of mice you'll need to discard for disease. Then plug in the values and calculate NY, the number of young mice to buy, and NO, the number of old mice to buy.
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Tenth Principle: Don't misinterpret artifactual correlations due to age effects. |
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Summary |
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• Make sure they're SPF, and free of the most obvious tumors and other significant diseases.
• Try not to pool unless absolutely necessary.
• Don't put clean SPF rodents into a conventional colony; the power of prayer, though redoubtable in some earthly domains, does not always fully prevent infections in rodents.
• Shake the inbred habit—this is not your father's rodent. Pick F1s if you have to use genetically homogeneous stocks, and make sure to confirm your key findings in multiple strains.
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Acknowledgments |
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Received April 9, 1999
Accepted August 25, 1999
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References |
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First Principle: Don't use...
