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Everything Wrong with Mouse Studies (Kinda)
Odors, magnetic fields, and even a mouse's siblings can all impact science.
“These creatures you call mice, you see, they are not quite as they appear. They are merely the protrusion into our dimension of vastly hyperintelligent pandimensional beings.”
— Douglas Adams, The Hitchhiker's Guide to the Galaxy
Viva la mice.
In 2009, a small group of Dutch biologists rounded up some lab mice, stabbed them with needles, and harvested their blood. Each drop was carefully collected into a small vial, and then spun in a centrifuge. As the tubes gyrated around fifty times per second, the blood separated; red cells split from white.
T-cells — a type of white blood cell that fights infections — were harvested from each vial, carried to a lab bench, and genetically modified. A gene encoding a protein, called a T-cell receptor, was added to each cell, thus re-programming them to detect and destroy tumors. The engineered T-cells were infused back into the rodents. A similar technique was being explored at the same time, on the other side of the Atlantic ocean, for cancer patients.
A few days later, though, alarms: Animals with the modified T-cells had massive spikes in their cytokine levels, a sign that their b
odies were under attack, crying out for help. The rodents grew weak; their bodies wasted away. After 14 days, most of the mice were dead. The scientists ended their paper with a warning: “It can be expected that the pathology seen here will occur within the clinic” — a.k.a. in real people, with real cancer, with real families.
Fortunately, that never happened. When the paper was published, surgeons in the U.S. had already done much the same T-cell engineering on 106 patients at the National Cancer Institute. They had “seen no evidence of [graft-versus-host disease] in any patient,” wrote surgeon Steven A. Rosenberg in a response letter entitled, “Of Mice, Not Men.”
Many scientific results, first observed in mice, never materialize in people. Sometimes that’s good. Most of the time, it’s bad. At this point, mice have been cured of just about everything under the sun, including cancer and aging (to an extent). Mouse embryos have even been sculpted from stem cells; no sperm, eggs, or uterus required. None of these advances, apart from some cancer studies, have worked out in humans. Many never will. (The hype from journalists shall continue evermore.)
A swelling tide of data now suggests that there are dozens of reasons why mouse studies fail to replicate, or why experiments don’t pan out in humans. Many of them sound absurd: The physical location of a cage and even the sex of a scientist have been shown, in some cases, to change a mouse’s behavior or physical size. After reading dozens of papers on this topic, I can’t help but feel that I wasted part of my life injecting chubby mice with drugs.
Mice and rats account for 95 percent of all animals used in biomedical research. More than 120 million mice are apparently killed each year in the name of science. For every 5,000 drug compounds tested in mice, five move into human studies. The ALS Therapy Development Institute, located just ten minutes from my home in Cambridge, once tested more than 100 potential drugs to slow ALS progression in mice, all of which had been reported to be helpful in other studies. None of them were beneficial, and “eight of these compounds ultimately failed in clinical trials, which together involved thousands of people.”
A recent article in The New Yorker suggests a solution to the problem. Instead of using caged mice for scientific studies, it argues, mice should be allowed to run free in open pastures. (Free-range mice?) With grass between their toes, mice would be less stressed and more attuned to their natural rhythms. Preliminary studies suggest this is a good idea. From the article (added links my own):
“At the University of Utah, the biologist Wayne Potts unleashed some of his lab mice into barn-like structures, where they can socialize and mate; so far, his cage-free mice have accurately predicted the health effects of high-fructose corn syrup, the statin Baycol, and the antidepressant Paxil. (They failed to predict side effects to a discontinued arthritis drug, Vioxx.)”
I like reading The New Yorker. I aspire to its witty, conversational, I-am-telling-you-about-this-thing-as-if-we-are-friends tone. But I was also disappointed in the article for a trivial reason: It doesn’t link to much of anything. And so, like the committed writer I am, I went out and searched for all the bullshit reasons that mouse studies could fail to replicate. The list that follows is by no means exhaustive; I deliberately chose papers and articles with strange outcomes.
Let Me Count the Reasons…
The Bible of mouse caretaking is called the Guide for Care and Use of Laboratory Animals. It did not change at all between the years 1972 and 2019, despite everything I’m about to tell you.
Mice raised at five different animal facilities in Europe, under otherwise identical conditions, have “persistent differences in body weight” and behavior. There are even differences in how their genomic DNA is packaged inside of neurons. Nobody really knows why.
If two different scientists at the same university carry out the same experiment on mice, their results will be more replicable than the same experiment, carried out by the same person, at separate universities.
Mice that give birth in cages with little toys or knick-knacks produce more pups. Those pups are larger after 21 days.
“Mice housed on deep bedding had smaller adrenal, kidney, liver and heart weights as well as larger body and tail lengths compared with groups kept on shallow bedding” after just 12 weeks, according to one study.
Mice handled by male scientists feel less pain. The finding holds true when a female scientist does the experiment but holds a t-shirt, previously worn by a man, near the mouse. The effect fades away after 30 minutes.
Mice spend less time licking an irritated part of their body when a human is nearby, “even if that ‘person’ is a cardboard cutout of Paris Hilton.”
Animals stored on higher shelves are more stressed and have impaired immune systems, probably because these areas are closer to lights and vibrate more.
Mice on the top shelf of a rack receive 20-80x more light than mice housed at the bottom.
Mice exposed to even dim light during the night (e.g. an LED on a computer monitor) “had a body mass gain…about 50 percent more than other mice that lived in a standard light-dark cycle.”
After just four weeks, mice exposed to a dim light during the night ate more than those in complete darkness. (Mice, like humans, raid the proverbial refrigerator when they can’t sleep.) Many genes linked to inflammation were also activated.
Mice kept in cages with wood chip bedding eat about 1.5 grams of their mattress every day. This changes the bacteria in their microbiomes.
Historically, male mice were used in research six-times more often than females because they don’t have an estrous cycle, and their metabolisms were therefore thought to be more predictable. About 80% of drugs are tested only on male mice. Some drugs, notably Ambien, are more potent in females and cause more side effects.
A study, published last week, suggests that female mice are less erratic than males while exploring an open space. The estrous cycle of female mice shows only “a very weak effect on their behavior.” Other scientists have said much the same thing since at least 2018.
The body temperature and activity patterns of male mice vary more in “a day than females do across an entire estrous cycle.”
About 6% of all mouse genes are regulated in sex-specific ways. The expression level of more than 1,000 genes varies between males and females, and the level of another 600 genes wobble, up and down, during a female’s estrous cycle.
The genes turned on in a specific type of immune cell, called a neutrophil, also differ between males and females.
Grain-based food usually contains unknown amounts of phytoestrogens, which change the onset of an animal’s puberty.
The standard diet for mice, called AIN-93, hasn’t changed in 30 years. But manufacturing of that food does: Even if you use “the same grain-based diet used in the past by others, its composition will likely differ.”
The sexes of a mouse’s siblings can skew an animal’s behavior. “Female-skewed litters demonstrated more social play, while the male-skewed litters demonstrated more solitary play.”
Rooms with a higher humidity are associated with lower pain thresholds for mice scorched with hot water. Nobody really knows why.
Most animal facilities change the bedding for mice every week, which causes “heart rate, blood pressure, and locomotion in both male and female mice” to spike for between 75 - 105 minutes. This effect persists even after mice have been moved many times.
Mice exposed to a regular, 37 Hertz magnetic field spend less time exploring open spaces, and more time sleeping.
Mice are kept in rooms between 69 and 79 degrees F. “But the natural comfortable temperature for mice is warmer — between 30 and 32 degrees Celsius (86 to 90 degrees Fahrenheit).” Colder mice experience more stress, their tumors grow faster than mice kept in warm rooms, and “mice genetically modified to develop obesity only gained a lot of weight at warmer temperatures but not at colder temperatures.”
Keeping mice at higher temperatures also blunts their muscle gain after exercise.
Two mice of the same strain are often genetically distinct, even if they’re labeled as isogenic. DNA differences accumulate over generations of breeding.
The microbiome of an animal can influence the effects of a drug. In one instance, a lab at Michigan State University was “testing how a certain drug affects bone density, and they found that treated lab mice lost bone compared with controls.” When they repeated the experiment on identical mice, of the same strain and from the same vendor, those mice gained bone density. A third experiment found no effect. Each animal had a different microbiome.
The FDA used to require animal tests before a human drug trial could proceed; they reversed that decision in January. As calls for more reproducible mouse studies continue to grow, many scientists have turned to organoids for experiments instead; organ-specific tissues (mini-brains, mini-hearts, mini-livers) derived from stem cells.
But all is not lost for mice. Some of these confounders are easy to control — Use more females! Turn up the temperature! Turn down the lights! Others are strange and probably impossible to solve — I mean, how do you correct for male body odors? I can only shower so often!
Despite their drawbacks, mice are still the best we have for biomedical research. The mouse and human genomes are about 85% identical in protein-coding genes. More than a hundred years of scientific history is based on this animal alone. We owe our long lives and abundant medicines to mice.
Now, before I end this essay, I want to tell you a story.
Every Sunday for four years of my life, I hopped on a bicycle and pedaled over the Iowa River to a gothic building, called the Medical Laboratories. Built in 1928, its hallways were lined with sharp fluorescent lights and peeling rubber floors. While my friends were busy getting drunk outside the football stadium, I donned a mask and took an elevator up to the attic. The smell of mice pierced my nostrils before the doors even opened. After arriving at my destination — a large steel door, plastered with warnings — I would put on my lab coat, pull on my gloves, and stretch cotton booties over my sneakers.
The “Mouse House” — as I called it — was filled with dozens of rooms, but my animals were kept in the first room to the right. And it was here, every Sunday, that I would pull my chubby mice from their shelves, one cage at a time, and inject them with drugs. My goal, ostensibly, was to prevent heart attacks. Of course, I never achieved that.
But I did achieve something else: A deeper appreciation for these animals. Spending four years in a smelly room, with stressed-out mice, will change your outlook on things. It will teach you to be patient, sympathetic, and gentle. Each time I picked up a mouse by the scruff of its neck and gently slid a needle into its belly, I’d whisper under my breath, “Sorry, sorry, please don’t be mad at me.” No mouse ever bit me — maybe they knew I was apologetic? — but many of them did look me in the eye. I often saw a glint of fear there, and also a sign of something else; I’d say intelligence.
In The Hitchhiker’s Guide to the Galaxy, Douglas Adams writes: "Earthman, the planet you lived on was commissioned, paid for, and run by mice."
Swap “planet you lived on” with “entirety of modern medicine,” and you’ll see these animals as I have.
Thank you to my wife, Claudia, for making this essay possible.
The views expressed in this blog are entirely my own and do not represent the views of any company or university with which I am affiliated.