Going
without sleep for too long kills animals but scientists haven’t known
why. Newly published work suggests that the answer lies in an unexpected
part of the body.
Feeling dead tired? Scientists may finally be on the verge of learning why too little sleep is inevitably fatal.
Inside
a series of tubes in a bright, warm room at Harvard Medical School,
hundreds of fruit flies are staying up late. It has been days since any
of them have slept: The constant vibrations that shake their homes
preclude rest, cling as they might to the caps of the tubes for respite.
Not too far away in their own tubes live other sleepless flies,
animated with the calm persistence of those consigned to eternal day. A
genetic tweak to certain neurons in their brains keeps them awake for as
long as they live.
They do not live long. The shaken flies and the engineered flies both
die swiftly — in fact, the engineered ones survive only half as long as
well-rested controls. After days of sleeplessness, the flies’ numbers
tumble, then crash. The tubes empty out. The lights shine on.
We all know that we need sleep to be at our best. But profound sleep
loss has more serious and immediate effects: Animals completely deprived
of sleep die. Yet scientists have found it oddly hard to say exactly
why sleep loss is lethal.
Sleep is primarily seen as a neurological phenomenon, and yet when
deprived creatures die, they have a puzzlingly diverse set of failures
in the body outside the nervous system. Insufficient sleep in humans and
lab animals, if chronic, sets up health problems that surface over
time, such as heart disease, high blood pressure, obesity and diabetes.
But those conditions are not what slays creatures that are 100% sleep
deprived within days or weeks.
What does sleep do that makes it deadly to go without? Could
answering that question explain why we need sleep in the first place?
Under the pale light of the incubators in Dragana Rogulja’s lab at Harvard Medical School, sleepless flies have been living and dying as she pursues the answers.
On a cold morning this winter, Rogulja leaned over a tablet in her
office, her close-cropped dark hair framing a face of elfin intensity,
and flicked through figures to explain some of her conclusions. Rogulja
is a developmental neuroscientist by training, but she is not convinced
that the most fundamental effect of sleep deprivation starts in the
brain. “It could come from anywhere,” she said, and it might not look
like what most people expect.
She has findings to back up that intuition. Publishing today in the journal Cell,
she and her colleagues offer evidence that when flies die of
sleeplessness, lethal changes occur not in the brain but in the gut. The
indigo labyrinths of the flies’ small intestines light up with fiery
fuchsia in micrographs, betraying an ominous buildup of molecules that
destroy DNA and cause cellular damage. The molecules appear soon after
sleep deprivation starts, before any other warning signs; if the flies
are allowed to sleep again, the rosy bloom fades away. Strikingly, if
the flies are fed antioxidants that neutralize these molecules, it does
not matter if they never sleep again. They live as long as their rested
brethren.
The results suggest that one very fundamental job of sleep — perhaps
underlying a network of other effects — is to regulate the ancient
biochemical process of oxidation, by which individual electrons are
snapped on and off molecules in service to everything from respiration
to metabolism. Sleep, the researchers imply, is not solely the province
of neuroscience, but something more deeply threaded into the
biochemistry that knits together the animal kingdom.
More Fatal Than Starvation
The first studies to investigate total sleep deprivation had a maniacal quality
to them. In Rome in 1894, Maria Mikhailovna Manaseina, a Russian
biochemist, made a presentation at the International Congress of
Medicine about her experiments on 10 puppies. She and her lab assistants
had kept the dogs awake and in constant motion 24 hours a day; within
about five days, all the puppies had died. Sleep deprivation seemed to
kill puppies much more quickly than starvation, she reported: “The total
absence of sleep is more fatal for the animals than the total absence
of food.”
Autopsies revealed that the puppies’ tissues were in bad repair,
particularly in the brain, which was rife with hemorrhages, damaged
blood vessels and other gruesome features. Sleep, Manaseina concluded,
is not a useless habit. It does something profound for brain health
.
More all-day, all-night dog walking followed. In 1898 Lamberto Daddi,
an Italian researcher, published detailed drawings of the brains of
dogs that had been sleep-deprived; he reported apparent degenerative
damage in the brain, similar to that seen in dogs that had faced other
stressors. Around the same time, the psychiatrist Cesar Agostini kept
dogs in cages rigged with bells that jangled horribly whenever they
tried to lie down and sleep, and in the 1920s researchers in Japan did
something similar with cages studded with nails.
The studies, aside from their consistent cruelty, had a similar
weakness: They had no valid controls. The dogs had died and their
tissues looked abnormal — but was that truly because they had not slept?
Or was it because nonstop walks and stimulation are inherently
stressful? Separating the effects of sleeplessness from being kept on
your feet until it killed you seemed impossible.
The Turntable Cage
It took decades for scientists to return to the question in a serious way. In the 1980s, Allan Rechtschaffen, a sleep researcher at the University of Chicago celebrated for his pioneering work on narcolepsy, began to design experiments
that could separate the effects of overstimulation from those of
sleeplessness. He devised a rat cage in the form of a turntable
suspended over water. A divider ran down the middle, so animals could
live on either side while the turntable floor beneath them spun freely.
Into the device the experimenters put pairs of rats, one of which was
destined to be denied sleep. Whenever that rat tried to rest, the
scientists spun the table, knocking both rats into the water.
This setup ensured that although both rats fell into the water
equally often, the control rat could still catch some winks whenever the
rat denied sleep was active. In fact, control rats managed to sleep
about 70% as much as they normally would, suffering only mild sleep
deprivation. The unluckier experimental rats got less than 9%, almost
total sleep loss.
Both sets of rats were disturbed the same number of times. Both
suffered the stress of falling into the water and having to clamber back
out, dripping. But only the severely sleep-deprived rats began to
decline. Their fur grew rough and disheveled, and it went from white to a
mangy yellow. They developed lesions on their skin. They lost weight.
After around 15 days on average, they died. Rechtschaffen had discovered
a way to show that sleep loss itself really did kill.
For the graduate students running these experiments, the days were
long. “The lab was in an apartment building, so you’d have a bedroom
next to an animal testing room,” said Ruth Benca,
a professor of psychiatry at the University of California, Irvine who
worked with Rechtschaffen for some years. “They had bedrooms next to the
rooms where their animals were being deprived so they could monitor
around the clock.”
The work was challenging in other ways as well. “They were tough,
tough experiments to do, psychologically, to put an animal through
that,” said Paul Shaw,
one of Rechtschaffen’s later graduate students and now a professor of
neuroscience at Washington University in St. Louis. “The last seven days
of the experiment, you’re working with this cloud over your head.” When
his rats were just a day or two from death, the experimental protocol
called for him to let them sleep and observe their
electroencephalograms, or EEGs. Shaw recalls that as the monitor
exploded with life, announcing the animals’ long-awaited slumber, he
felt a weight fall from his shoulders. “To this day I can see it,” he
said, speaking of the EEG readout. “I could put it in a frame up on my
wall, and it could make me happy every time.”
But the work was also thrilling. “You have to believe in the outcome
to do it. There’s no other way,” Shaw said. He arrived at the lab after
students who had pioneered these experiments received their degrees and
left, but he still heard their stories at meetings, where they
reminisced about the excitement. “No one wanted to get their Ph.D.,” he
recalled, because if they could stay, “they all thought that tomorrow,
they’d discover the function of sleep.”
Confusing Causes of Death
Rechtschaffen’s experimental successes should have finally enabled
scientists to see how insufficient sleep kills, which might have led to
bigger insights into what makes sleep so indispensable. But when the
researchers performed autopsies on the animals, what they found mostly
just added to the confusion. There were few consistent differences
between the control rats and those that died from lack of sleep, and no
sign of what had killed them. The deprived rats were thin and had
enlarged adrenal glands, but that was about it. “No anatomical cause of
death was identified,” the researchers concluded.
Observations of the animals’ behavior showed something more
interesting. “Animals [chronically] sleep-deprived under these carefully
controlled conditions would increase their food intake two and three
times normal amounts, and lose weight,” said Carol Everson,
a professor of medicine and neurobiology at the Medical College of
Wisconsin who was one of Rechtschaffen’s graduate students. “We did all
sorts of metabolic studies to try to find out if there was an impairment
we could detect.”
There was a strong feeling in the sleep field, however, that answers
about sleep’s most basic functions would be found in the brain. John Allan Hobson, a prominent Harvard Medical School sleep researcher, had just published a paper in Nature with the title “Sleep is of the brain, by the brain and for the brain.” As Shaw recalled, “This captured the zeitgeist of the entire sleep community.”
Indeed, the vast preponderance of sleep research today still centers
on the brain and on subjects like cognitive impairment. Sleep loss does
alter metabolism in humans — there are connections to diabetes and
metabolic syndrome — but public health researchers are often the only
ones who concern themselves with it. Those looking to understand the
fundamental purpose of sleep rarely seek answers in metabolism or other
chemical processes.
Reactive Oxygen Species
The neurons involved in regulating sleep are a focus of Rogulja’s
work. But the fact that sleep loss impairs circulation, digestion, the
immune system and metabolism made her curious about whether these were
downstream effects of neurological problems, or if they were
independent. “It seems like it can’t be all about the brain,” she said.
She knew about the Rechtschaffen experiments — “real classics” — and
that there had been few follow-ups. Once it was established that total
sleep loss kills, using deprivation to study sleep’s purpose had fallen
by the wayside. In the intervening decades, however, fruit flies had
become a major model organism in the sleep field, because their genetics
are widely understood and easy to manipulate and they are inexpensive
to keep in the lab. Many sleep discoveries first made in flies have been
verified in mammals. With the rise of flies as proven test subjects,
when Rogulja became curious about terminal sleep deprivation, it again
seemed like a plausible thing to study.
When the postdoctora researcher Alexandra Vaccaro
arrived at Rogulja’s lab in 2016, the two came up with a plan. First,
from other laboratories they obtained flies genetically engineered to
have temperature-sensitive channels in certain neurons. Above 28 degrees
Celsius, the channels opened and stayed open, keeping the neurons
activated and the flies awake. With the channels closed, the flies
enjoyed normal 110-day life spans. With the channels open, they started
dying of total sleep deprivation after only 10 days or so, and they were
all dead within 20 days.
