NEWS FEATURE

What happens when lab animals go wildExperiments on mice living in more natural habitats can deliver results dramatically different

from those in traditional laboratories—with profound implications for biomedical science.

Carolyn Beans, Science Writer

In the summer of 2015, a pioneering band of labora-tory mice did something their ancestors hadn’t donefor roughly 1,000 generations—they went outside.

It was hardly a trek into the wilderness. The 90 micewere fenced into pens, with feeding stations providing allthe mouse chow they could eat and aluminum pie platesdangling over their heads to deter passing hawks. Still, itwas aworld away from their former home in the laboratoryof Andrea Graham, an ecological and evolutionaryimmunologist at Princeton University. These mice couldnow roam around an area of roughly 180 square meters,feeling the dirt under their feet and rain on their backs.

Graham’s project aimed to show how a mouse’snatural environment affects its susceptibility to parasiticworms called nematodes, which live in the animal’s di-gestive tract. As with so many other conditions, from

cancer and diabetes to Alzheimer’s disease and stroke,scientists study nematode infestations in mice to de-velop treatments for humans. Most of these studiestake place in laboratory settings where researcherscan control myriad complicating variables such as tem-perature, diet, and social interactions.

But researchers such as Graham are questioningwhether this tactic is always the best approach. If thenatural environment of a mouse—or a human—is itselfa major factor affecting a disease or its treatment,studying it under strict lab conditions could skew theresults. As pressure mounts for scientists to makemouse findings translatable to humans, a small butgrowing number of researchers are designing studiesthat use more natural experimental conditions. Theiraim is not to replace traditional lab studies but ratherto complement them with real-world context.

Some studies, for example, have already shown thatexperiments on mice in a barn-like setting can uncovera drug’s potential side effects, which traditional preclin-ical research had missed. And Graham’s results fromthe nematode study, which used a nematode-resistant strain of mouse, were “just eye-popping,”she says. A few weeks after she infected both indoorand outdoor mice with nematodes, the outdoorgroup harbored massive infections, with a mass ofworms 100 times greater than the indoor group (1).

Researchers have good reason to establish acontrolled environment, long a hallmark of scientificrigor. And as some start to bring their lab animals intowild territory, they grapple with many questions: Whatis the best way to mimic the wild? How shouldresearchers monitor animals without disturbing theirenvironment? And how much control can they reallyafford to give up before experiments become irrepro-ducible or logistically and financially intractable? Onething is certain: Take mice out of the cage and theresults can be fundamentally different.

Translation TroubleMice are still the human stand-in of choice in bio-medicine. The vast majority of their genes have acounterpart with the same function in humans, and

Andrea Graham of Princeton University moved lab mice outdoors to test theirsusceptibility to nematodes in a more natural setting. Image courtesy ofDavid Tricker (photographer).

Published under the PNAS license.

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they suffer from many of the same diseases. Re-searchers can manipulate mouse genomes to emulatethe human conditions. And they can make experi-ments more repeatable using inbred strains—lineagesof genetically identical mice created through 20 or moregenerations of mating between sisters and brothers orparents and offspring (2).

Yet most successful therapeutic treatments inmice do not translate successfully into humans (3).“The ultimate goal is translatability,” says laboratoryveterinarian Norman Peterson at MedImmune inGaithersburg, MD, who serves as a board member ofthe North American 3Rs Collaborative, an organiza-tion dedicated to replacing, reducing, and refiningthe use of laboratory animals. “If an animal is in anunnatural environment and stressed and hormonesand cytokines are out of whack, the question is: Isthat the best model? In most cases it is not.”

In some ways, the life of a lab mouse is unnaturallystressful. Many argue, for example, that the standardtemperature for mouse facilities (20–26 °C), the sub-ject of one of several continuously updated recom-mendations contained in theNational Research Council’sguide to lab-animal care and use (4), is comfortable forresearchers but lower than optimal for mice (5, 6).

In other ways, the life of a lab mouse is unnaturallycushy—no competition for territory and all the foodthey can eat. “Most organisms function better undersome level of eustress, the good stress,” says labora-tory animal veterinarian Kathleen Pritchett-Corning ofHarvard University in Cambridge, MA. “When yourespect the animal’s natural behaviors, you get thebest results out of those animals.”

Biologist Wayne Potts of the University of Utah inSalt Lake City has been encouraging natural behaviorin his study animals since the late 1980s. As apostdoctoral fellow at the University of Florida, Pottswanted to understand how natural selection and sex-ual selection affect the major histocompatibility com-plex (MHC), a collection of genes that helps theimmune system identify bacteria and other foreignsubstances. He built a barn-like structure to housewild strains of mice so he could watch these forcesplay out naturally, and he found that female micepreferred males with MHC genotypes different fromtheir own (7). But he also realized that the “mousebarn” offered a powerful way to study mice undermore natural conditions.

Since then, Potts has conductedmany experimentsthat he calls organismal performance assays (OPAs) instructures that function much like barns. He usesstrains of genetically diverse house mice (Mus mus-culus) that breed outside of family groups, unlikeclassic inbred lab strains. Potts typically puts about30 of these mice into 32-square-meter enclosures thatare divided into territories by sheets of metal mesh.Deluxe territories boast a feeding station and a plasticbin for hiding, while less desirable areas lack thehideout. The mice can easily climb between territories,and males compete with each other up to 50 times aday to defend or win territories. Those who win territoryalso win mates.

After putting mice from a treatment group and acontrol group together in the barn, Potts judges thehealth effects of the treatment by the number of off-spring sired by male mice from each group. “If theexperimental manipulation reduces health, then ingeneral they’re going to be less able to maintain ter-ritories, and so have fewer offspring,” Potts explains.

Results, ReinterpretedIn one case, Potts used an OPA to study the healthimpacts of sugar consumption (8). Previous studieshad only found disease outcomes when lab animalswere fed on diets containing far more sugar than hu-mans typically ingest. What, Potts wondered, mightbe the outcome with more modest amounts? So for26 weeks, he fed mice a diet that reflected what manyAmericans actually eat, with a quarter of their caloriescoming from fructose and glucose. When he sub-sequently put the mice in his barn-like enclosure, theeffect was stark. The death rate of sugar-fed femaleswas almost twice as high as those in the control groupwho had eaten a sugar-free diet. Meanwhile, sugar-fed males won fewer territories and produced one-quarter fewer offspring than control mice. The resultsstrongly suggest that sugar in these amounts is bad formammalian health, although the experiment could notexplain exactly why.

This barn-based approach can also flag drug risksthat traditional preclinical and clinical trials miss. Pottsfound that the statin drug, cerivastatin (Baycol), whichwas pulled from the market after being linked toskeletal muscle breakdown, caused males to produce41% fewer offspring and females to produce 25%fewer offspring compared with control mice (9)—consequences that were not evident when researchersconducted traditional tests in lab animals (10).

Wayne Potts judges the health consequences of a treatment largely by its effecton how many offspring male mice produce. The mice compete for territories andmates in barn-like structures such as this one at the University of Utah. Imagecourtesy of Douglas Cornwall (University of Utah, Salt Lake City, UT).

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But the barn is not foolproof. In a test of the recalledantiinflammatory drug, rofecoxib (Vioxx), Potts’ experi-mental design was no more sensitive to the increasedrisk of cardiovascular events than traditional preclinicaltests (11). That may be because of sample size: Pottsestimates that his study would have needed 9,000miceto pick up a signal of the rare events.

In another series of studies, Potts used his mousebarn to explore a key question in genetics: Why is itthat 10 to 15% of mouse genes can be disruptedwithout having any noticeable effect on the mouse?One possible explanation is that many genes code forsimilar functions, so if researchers delete one, anothertakes over the job. Indeed, Potts’ colleagues at theUniversity of Utah, Mario Capecchi and Petr Tvrdik,now at the University of Virginia in Charlottesville, hadfound that two genes called Hoxa1 and Hoxb1, whichhelp control brain stem development, were function-ally redundant—replacing Hoxb1 with Hoxa1 had noeffect on mice in the lab (12). But because they stud-ied lab animals in cages, they could only check formajor effects on traits such as locomotion and bodysize. “The subtle behavior alterations are impossibleto pinpoint,” says Tvrdik. So the pair teamed up withPotts and discovered that in the barn males with thisgenetic manipulation produced 64% as many offspringas controls (13). Replacing Hoxa1 with Hoxb1 hadsimilar effects (14).

“We like to think of the organismal performanceassay as a high-throughput initial health screen,” saysPotts. “We can detect most insults to health withouthaving a priori knowledge of what the mechanismmight be.”

A Spectrum of WildnessUnlike Potts, Graham studies how traditional strainsof inbred lab mice fare in wilder conditions. Asidefrom her “eye-popping” study on nematode-resistantmice, she also studied a mouse strain that’s commonlyused as a model for nematode susceptibility, becauseit lacks the immune response necessary to expel theparasites. In the field, the susceptible strain and theresistant strain suffered from nematodes equally.“Between 10 and 20 days outdoors made that massivedifference between the mouse genotypes prettymuch go away,” she says.

Several factors could be responsible for this dra-matic effect. Bacteria were already known to play acritical role in helping nematode-egg hatching (15),and “microbial diversity went through the roof” in heroutdoor mice, says Graham. Secondly, fighting off anematode infection requires the right sort of immuneresponse. Unlike those in the lab, outdoor mice in-fected with nematodes tended to have elevated levelsof the T cell Th1, known to be more effective atfighting off bacteria and viruses, rather than the T cellTh2, which is typically better at fighting off worm in-fections. Regardless of the underlying mechanism,Graham’s findings show that the environment plays amajor role in nematode susceptibility—hence findingsin the lab may not translate into the clinic.

Other researchers are taking smaller steps towardmore natural conditions but still seeing dramatic ef-fects. For example, David Masopust of the University ofMinnesota in Minneapolis studied the effects of mixingpet-store mice with lab mice from pathogen-free facil-ities. Lab mice that survived exposure to their dirtyroommates acquired immune systems with a greaterabundance and variety of T cell types, more closelyresembling the immune systems of adult humans (16).

Pritchett-Corning argues that such results arepushing science toward a paradigm shift in experi-mental design (17) and that Potts’ OPA approach is akey step in the right direction. “He’s turned the wholething on its head and looked for translational and ac-tual biological effects, rather than regarding mice asthe furry test tube,” she says.

Potts believes it will take a cultural shift for bio-medical researchers to consider conducting studies inmore natural arenas. One big reason why: Researchersoften focus on nailing down the molecular mecha-nisms underlying a disease, whereas the mouse barn ismost useful for showing effects at the level of thewhole mouse. Yet Potts contends the approaches arecomplementary. Once the barn raises a red flag abouta treatment, researchers can use traditional tech-niques to search for the underlying molecular basis.Or, Graham suggests, researchers might also developtreatments in a controlled lab and then test them in amore natural environment.

Of course, there are good reasons why researcherstypically stick to traditional laboratory settings. “Theessence of research is often being able to control theparticular variable that you are studying,” notes neu-ropharmacologist James Barrett of Drexel University inPhiladelphia, who is the editor-in-chief of the Hand-book of Experimental Pharmacology. “You can reallyonly do that under highly controlled conditions whereyou know the strain of the animals, the conditionsunder which they exist, their diet, the daylight cycle,the humidity in the room—all of those variables areknown to exert sometimes subtle and sometimesprofound effects on what it is you are studying.” Letgo of some of this control, and it becomes more dif-ficult to repeat the experiment and produce the sameresults, Barrett notes.

But reproducibility, while important, can come atthe cost of relevance when studies are conducted un-der narrow experimental conditions on inbred strains ofmice, counters surgeon and medical researcher RonaldTompkins, founding director of the Center for Surgery,Innovation and Bioengineering at Massachusetts Gen-eral Hospital in Boston. “The human is way morecomplex—by genetic diversity, and by environmentaldiversity,” he notes. In general, he argues that re-searchers studying inbred mouse populations shouldoffer a caveat in their publications: wilder settings andoutbred mice could yield different results.

Tompkins says researchers studying geneticallydiverse mice under more natural conditions are on theright track. But he contends that attempts to applymouse findings to humans deserve even more scrutiny.In 2013, he and colleagues presented a cautionary tale,

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a controversial study suggesting major shortcomingswhen applying mouse-model genomics to human in-flammatory disease (18). “Mice aren’t men,” he says.

Making Better ModelsThose researchers who do embark on seminaturalexperiments face a suite of logistical challenges thattheir institutions may not be ready to accommodate.“Universities have great animal facilities for the mostpart, but they are not prepared to build barns forpeople. It requires a big push on the end of the in-vestigator,” says evolutionary geneticist Beth Dumontof the Jackson Laboratory in Bar Harbor, ME. Space isone of the biggest challenges. “To build a seminaturalenclosure, you’re going to take a room that could haveheld a couple of hundred to 1,000 mice down to 40,”says Pritchett-Corning.

Dumont says that classic inbred strains of mice arealso much easier to work with than wild mice or eveninbred mouse strains whose ancestors were collectedfrom the wild only about 20 generations back. Theseclassic strains are less jumpy, easier to handle, andreproduce readily in the lab. “Handlers have in-advertently selected for them to be docile,” she says.

It’s also difficult to administer separate treatments todifferent groups when they are all in the same enclo-sure. Potts calls the results from his sugar study “con-servative” because both his control and sugar-fedmice,once transferred to the barn after their initial feeding, allhad the same high-sugar diet, drawing from feedingstations on their own. “Wehad, and still have, no way tokeep the mice on their respective diets when they areactually doing the competition in the barn,” he says.

Looking to investigate the effect of a more natu-ral environment on the brain, Graham’s colleague,Princeton neuroscientist Elizabeth Gould, encounteredher own difficulties. Gould, who explores how the adultmammalian brain produces new neurons in response todifferent stimuli, studied a group of mice she borrowedfrom Graham’s nematode research. “We were very in-terested in what kinds of changes in brain growth wewould see in animals that were living outside in anenriched environment, with stresses like needing to foragefor food and deal with weather,” she says. Gould foundthat outdoor mice had more new neurons compared withmice kept under standard laboratory conditions, resultsthat she plans to publish soon. But to conduct standardcognitive and anxiety tests on the animals, Gould had tocapture them and bring them back into her lab—astressful episode for the mice that could itself affect theresults. “It didn’t make the findings useless, but it’ssomething that you have to keep in mind,” she says.

Even in the most well-crafted experimental designs,the mice may not experience all the environmentalvariables that could affect disease. And in many cases,researchers are still exploring which of many naturalvariables—from microbes and weather to diet and so-cial interactions—actually really matter for a study. “It’searly days,” says Graham. “Maybe in another 5 or10 years there will be a succinct statement we canmakeabout the important differences between lab and field.”

“We’re not saying that mouse barns are going tosolve every problem,” adds Potts. “Just that theywill solve many that the conventional methodsare missing.”

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3 Rutkin A (October 26, 2016) Lab mice are sending us on a wild goose chase. New Scientist. Available at https://www.newscientist.com/article/mg23230971-300-lab-mice-are-sending-us-on-a-wild-goose-chase/. Accessed November 21, 2017.

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8 Ruff JS, et al. (2013) Human-relevant levels of added sugar consumption increase female mortality and lower male fitness in mice.NatCommun 4:2245.

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