Human limits: Why not sleep leads to death?

Long periods of non-sleep can lead to animal death, but scientists can’t say exactly why, and new research suggests that the answer lies in an unexpected part of the body. In a bright, warm room at Harvard Medical School, hundreds of fruit flies are “staying up late” in a row of test tubes. They haven’t slept for days because these test tubes keep shaking, making them unable to rest and just try to rest on the lid of the test tube.

In other test tubes not far away, there are also many fruit flies that never sleep. The researchers fine-tuned the genes of some of the neurons in their brains to keep them awake while they were alive.

Of course, these fruit flies don’t live long. Both the fruit flies that vibrate at all times, or the genetically modified fruit flies, eventually die quickly. In fact, genetically modified fruit flies survive only half as long as the resting control group. After several days of sleep deprivation, the number of fruit flies dropped sharply until it died.

It is well known that we need sleep to maintain optimal physical and mental conditions, and severe sleep deprivation can have a more serious and direct effect: animals that are completely sleep-deprived in experiments die quickly. However, scientists have found that the problem of sleep deprivation is not as simple as thought.

Human limits: Why not sleep leads to death?

Dragana Rogorga, an assistant professor of neurobiology at Harvard Medical School, speculates that the most fundamental effect senile effects of sleep deprivation begin outside the brain and “probably come from anywhere.”

Sleep can fundamentally be regarded as a neurological phenomenon, however, when sleep-deprived animals die, there are a series of confusing functional problems outside the nervous system in their bodies. Humans and experimental animals who do not get enough sleep for long periods of time can develop a range of health problems over time, such as heart disease, high blood pressure, obesity and diabetes. But in fact, for animals that do not sleep at all for a few days or weeks, these diseases are not the cause of their deaths.

What is the important role of sleep, so that sleep deprivation leads to death? Does the answer to this question explain why we need sleep?

Dragana, a researcher at Harvard Medical School, is trying to find answers to these questions. In her lab, pale light shines on the incubator, and the “sleepless fruit fly” continues to move from survival to death in the test tube.

On a cold winter morning, Rogurga came to the office. She leaned in front of the tablet and began to use charts to explain some of the conclusions of the experiment. Although Rogurga is a trained developmental neuroscientist, she doesn’t think the most fundamental effect of sleep deprivation begins with the brain. “It could come from anywhere,” she said, and it probably didn’t look like most people expected.

Human limits: Why not sleep leads to death?

The researchers speculate that sleep deprivation may well be rooted in a group of highly active molecules called “reactive oxygen.” Significant increase in reactive oxygen levels can be found in sleep-deprived animal guts

Rogurga made some discoveries that supported his intuition. In a recent issue of Cell, she and colleagues provided evidence that when fruit flies die from sleep deprivation, the deadly changes occur not in the brain, but in the intestines. Microscopic images show bright fuchsia in the indigo-blue fruit fly’s small intestine, revealing the accumulation of molecules that can damage DNA and cause cell damage. Shortly after the beginning of sleep deprivation, these molecules appear without warning; It’s worth noting that if fruit flies ingest antioxidants that neutralize these molecules, it doesn’t matter if they don’t sleep anymore. They live as long as their restable counterparts.

These results suggest that one of the most basic functions of sleep may also be the basis of a range of other functions, namely, the regulation of ancient biooxidation reactions. In this process, the gain or loss of a single electron determines the function of the molecule, affecting everything from breathing to metabolism. Sleep is not just a problem in the field of neuroscience, the researchers note, but more deeply linked to biochemistry and a series of animal kingdoms.

More deadly than hunger.

The original experiment into complete sleep deprivation had a certain crazy nature.

In 1894, Russian biochemist Maria gave a report at the International Medical Congress in Rome about her experiments on 10 puppies. She and her assistant kept the dogs awake 24 hours a day and kept moving; Sleep deprivation is more likely to lead to puppy death than hunger, she reported, and “total sleep deprivation is more deadly for puppies than total deprivation of food.”

Autopsy results showed that the dogs had a poor tissue repair, especially in the brain, where bleeding, blood vessel damage and other terrible features were everywhere. Manasina concludes that sleep is not a useless habit and has a profound effect on brain health.

Human limits: Why not sleep leads to death?

Alexandra Vaccaro, a postdoctoral researcher at the Rogurga Laboratory, found that fruit flies can recover from severe sleep deprivation and have a normal life span. However, for a period of time, they are still very likely to die from resleep deprivation.

Since then, more researchers have begun experimenting with sleep deprivation with dogs. In 1898, Italian researcher Lamberto Daddi published a report detailing the brains of sleep-deprived dogs. He reported that the dogs had significant degenerative damage to their brains, similar to the symptoms they developed when faced with other stress. Around the same time, the psychiatrist Cesar kept the dogs in a cage with bells, which sounded terrible whenever they tried to lie down and sleep. In the 1920s, Japanese researchers conducted similar experiments with nail-covered cages.

In addition to being very cruel, these experiments had a similar flaw: no effective control group was set. After these dogs die, their tissues look abnormal, but is it really because they don’t sleep? Or is it because of the constant walking and the stress itself gives? It seems impossible to completely separate the consequences of sleep deprivation from the effects of standing until death.

Rotating Cage

It took scientists decades to get the problem serious again. Allen, a sleep researcher at the University of Chicago, known for his pioneering work on narcolepsy, began designing experiments in the 1980s to try to distinguish overstimulation from the effects of sleep deprivation. He designed a rat cage shaped by a turntable, suspended from the water. There is a partition in the middle of the cage that can place the rats on either side of it, and the turntable below them can rotate freely. The experimenters put the rats in pairs into the device and completely deprived one of them of sleep: whenever the rat tried to sleep, the researchers rotated the turntable and knocked both rats into the water.

Human limits: Why not sleep leads to death?  

When the researchers used antioxidants to offset the accumulation of reactive oxygen in sleep-deprived fruit flies, their life expectancy returned to normal. However, it is unclear whether this approach will weaken other effects of sleep deprivation

Although both rats fell into the water as frequently, this setting ensured that the control group of rats could still take a nap when the sleep-deprived rats were active. In fact, the control group had only 70 percent of the normal sleep and suffered from mild sleep deprivation, while the other rat slept less than 9 percent of the normal, almost completely.

Both groups of rats were disturbed the same number of times, both suffering from falling into the water and having to swim to survive. However, only rats with severe sleep deprivation began to weaken. Their hair became coarse and messy, from white to dirty yellow. They develop lesions on their skin and lose significant weight. After 15 days of sleep deprivation (average), the rats died. Richshafin’s approach proves that sleep deprivation itself does lead to death.

For graduate students who conduct these experiments, the time is long. “The lab is in an apartment building, and your bedroom is next to an animal lab,” says Ruth Benca, a professor of psychiatry at the University of California, Irvine who has worked with RichShafen for several years. “

In other ways, the job is challenging. “It’s a very, very, very difficult experiment psychologically,” said Paul Shaw, a former graduate student and now a professor of neuroscience at Washington University in St. Louis. “With a day or two to go before the rats died, the protocol called for them to sleep and observe their eGEs. Shaw recalls that when the readings on the monitor announced that the rat had gone to his long-awaited sleep, he felt the burden on his shoulders off. “I still remember the EEG reading to this day, ” he said of the EEG reading. ” “

The cruel work is equally exciting. “You have to believe that it will bear fruit. There’s no other way,” Said Paul Shaw. When he came to the lab, the first students to do these experiments had earned their degrees and left, but he would still hear their stories at the meeting and the excitement of their memories. “No one took a Ph.D. in mind, ” he recalls, because they all thought that if they could stay, “they might find the function of sleep the next day.” “

Chaotic cause of death

The success of Richsaffin’s experiment should have allowed scientists to finally discover how sleep deprivation causes death and gain a deeper understanding of the causes of sleep. But when the researchers performed autopsies on rats, they found that most of the results added to the confusion. There was little consistent and significant difference between the control group and the rats who died of sleep deprivation, indicating what caused their deaths. Sleep-deprived rats were light in weight and had a swollen adrenal gland, “unable to identify an anatomical cause of death”.

Observations of animal behavior reveal something more interesting. “Under these carefully controlled conditions, animals with long-term sleep deprivation increase food intake, two to three times their normal intake, and lose weight,” said Carol, a graduate student at Richsaffin and a professor of medicine and neurobiology at the University of Wisconsin School of Medicine. “

However, researchers in the field of sleep research have a strong hunch that the answer to the most basic functions of sleep will be found in the brain. At the time, John Allan Hobson, a prominent sleep researcher at Harvard Medical School, had just published a paper in the journal Nature, entitled “Sleep is all about the brain, dominated by the brain, and used by the brain.” “It captured the zeitgeist of the whole field of sleep research,” Xiao recalls. “

In fact, the vast majority of sleep research today still focuses on brain and cognitive impairments. Lack of sleep does alter the body’s metabolism — it has been linked to diabetes and metabolic syndrome — but public health researchers are often the only ones concerned, and those who want to understand the basic functions of sleep rarely seek answers in metabolic or other chemical processes.

Reactive oxygen

The neurons involved in regulating sleep are the focus of Rogorga’s work, but the fact that lack of sleep can damage circulation, digestion, immune system and metabolism makes her curious. Are these downstream effects of neurological problems or independent of each other? “It doesn’t seem like it’s all a brain problem, ” she says.

She was well aware of Richschaffin’s experiment and thought it was “very classic”, but there was little follow-up to it. After the idea that total sleep deprivation leads to death was established, the researchers largely stopped using sleep deprivation to study the function of sleep. In the decades that followed, however, fruit flies became the dominant model creatures in the field of sleep because their genes had been well studied, easy to operate, and not expensive to preserve in the laboratory. Many of the sleep phenomena first found in fruit flies have been confirmed in mammals. Thus, with the popularity of fruit flies as an experimental object, the effects of sleep deprivation seem to have once again become a promising research topic.

In 2016, Alexandra, a postdoctoral researcher, came to Rogurga’s lab and the two came up with a research plan. First, they obtained fruit flies from other laboratories that had been genetically modified to have temperature-sensitive channel proteins in some neurons. When the temperature is above 28 degrees Celsius, these channel proteins open and remain open, keeping the neurons active, i.e. keeping the fruit flies awake. In the case of the channel closure, the normal life of the fruit fly is 110 days, and as the channel opens, the fruit fly begins to die of complete sleep deprivation in about 10 days and all within 20 days.

Interesting patterns emerged when Vaccaro experimented. If the channel is closed on the 10th day to let the fruit flies sleep, they will recover, life is as long as the control group, but if sleep deprivation is performed after 5 or 10 days, the fruit flies will die: no matter what damage they cause in the initial sleep deprivation, they obviously have not been repaired. Before sleep deprivation again, fruit flies must be given normal sleep for 15 days before they die immediately.

Vaccaro performed an autopsy on fruit flies with different sleep deprivation levels and found that their tissues did not appear to be damaged, with one notable exception: their internal organs were filled with reactive oxygen ( ROS). This is a class of oxygen-containing molecules that are highly chemically active. Some reactive oxygen is produced by organisms during normal respiratory, metabolic, and immune defense sourcing, sometimes for specific functions and sometimes as by-products. However, if reactive oxygen is not removed by antioxidant enzymes, they become extremely dangerous because unbalanced oxygen strips electrons from DNA, proteins and lipids. In fact, a week after the fruit fly was deprived of sleep, reactive oxygen appeared, causing the markers of oxidation damage to soar, a sign of a cell crisis.

Reactive oxygen levels peak on the 10th day of sleep deprivation. When fruit flies start sleeping normally, their reactive oxygen levels take about 15 days to reach the baseline again, the same time fruit flies can withstand further sleep deprivation.

Rogorga and Vaccaro did not expect such a clear outcome within the first few months of the project. The answer came too easily, which immediately cast doubt on them. When Rogorga presented the preliminary data at a Pew biomedical scholars’ conference, the excitement of the participants made her a little nervous. “It’s never been like this,” she said. She is more inclined to be cautious about the results of the study.

So for the past three years, Vaccaro, Rogoga, and postdoctoral researcher Joseph have been studying the apparent link between reactive oxygen and sleep deprivation. They used a more traditional method to deprive fruit flies of sleep time — shaking test tubes containing them every two seconds — and then checked to see if reactive oxygen levels were associated with sleep deprivation levels. They did it. The team looked at fruit flies with genetic mutations that promote sleep or sobriety, in which sleep-deprived fruit flies found reactive oxygen in their intestines;

The strangest and most exciting moment of the project has arrived. The researchers believe that if the oxidation of reactive oxygen kills fruit flies, they should probably provide them with antioxidants. It may sound like a funny healthy food experiment, but Vaccaro found antioxidants known to be effective in fruit flies and fed them to sleep-deprived fruit flies. To the researchers’ surprise, the life expectancy of these fruit flies returned to normal levels. The same happened when the researchers raised the levelof of antioxidant enzymes in the gut seofa intestines (but apparently this was not the case when they raised the levels of antioxidant enzymes in the fruit fly’s nervous system).

“I can’t imagine anything happier in science,” Rogurga said of the summer. ‘They don’t just live, they look good. “

Vaccaro and lab technicians, as well as collaborators at Harvard’s Michael’s Lab, conducted a simplified version of the fruit fly experiment on mice. They kept the mice in a cage with a rotating rod and kept them awake for five consecutive days. It was found that the mice had reactive oxygen fluorescence in their internal organs.

Intestinal problems

In Paul Shaw’s view, the team’s new paper is very interesting. “It’s very exciting to see them harness the power of genetics, ” he said. ” “Also, it is difficult to control stressors in experiments. The new study avoids the problem because it uses both genetic and mechanical methods for sleep deprivation. “It’s great, it’s great… I admire it,” Shaw said. “

The significance of these findings has yet to be further explored. The researchers believe that sleep is essential for the body’s oxidation regulation, especially in the intestines, which can have widespread effects on the body. As Rogorga et al. wrote in their new paper: “Death can be prevented by a single means, which proves that the gradual failure of almost all major bodily functions is due to a common cause.” “For the fruit flies they studied, antioxidants were the only way.

The findings also coincide with a series of previous reports linking oxidation processes to lack of sleep, particularly in the study conducted by Carol Everson. While working in Richschaffen’s lab, Everson became interested in metabolism. She realized early on that although the brain is the manager of sleep, sleep is not just about neuroscience. In sleep-deprived rats, she observed signs of a weakening of the immune system and found bacteria in the sterile tissue. In 2016, she and colleagues reported that they had found oxidation in the liver, lungs and small intestine sized in sleep-deprived rats. After sleep deprivation, it is often found that inflammatory markers are found floating in the tissue, but the source has been unclear, Everson said. If the oxidation process gets out of control somewhere in the body, the resulting cell damage can lead to an inflammatory response.

Everson also found that sleep-deprived rats have intestinal leaks that release bacteria into their blood. But from the observations of Rogorga and his colleagues, the fruit fly’s intestines do not appear to have leaked. In the other tissues they examined, the level of reactive oxygen did not appear to increase. Although fruit flies sometimes eat more during sleep deprivation, their levels of reactive oxygen in their guts do not appear to have changed.

It is not clear how the results of the rat and fruit fly will be integrated. While these experiments show that reactive oxygen kills fruit flies, this does not mean that reactive oxygen can kill rats, says George O,, a sleep researcher at Imperial College London. A small study of sleep-deprived people has shown that the composition of the gut microbiome changes after lack of sleep. This is an interesting preliminary finding that reveals another link between sleep and the intestines.

However, perhaps the most pressing problem is that we still don’t know where reactive oxygen comes from and why it accumulates in the gut. What processes (metabolic or other processes) produce reactive oxygen? Does lack of sleep lead to excessive production of reactive oxygen? Or does lack of sleep interfere with the normal process of removing reactive oxygen? Why is reactive oxygen associated with sleep? Rogurga plans to explore these issues in future experiments.

Behind all this is the widespread effects of sleep on the body. Learning, metabolism, memory, and countless other functions and systems are all affected by sleep, which can be interesting if these effects are derived from fundamental changes in active oxygen. But even if reactive oxygen is one of the causes of sleep loss, there is no evidence that sleep’s cognitive effects are based on the same mechanism. Even if antioxidants prevent premature death of fruit flies, other functions of sleep may not be affected, or even if affected, for other reasons.

These sleep-deprived fruit flies and their fluorescent guts remind us that sleep is a systemic experience, not just a function of the brain and nerves. The death of a fruit fly may provide an answer to why sleep loss is fatal and explain the role that sleep plays in connecting the body’s different systems. Paul Shaw is interested in what will happen next in Rogoga’s lab. “This is a very important issue, ” he said. ” “

Currently, researchers are studying upstream biological pathways in which reactive oxygen accumulates in the gut and affects body function, and they hope the study will help develop new treatments in the future to counteract the negative effects of sleep loss. For many people, lack of sleep is part of life. “Many of us are in a state of chronic sleep deprivation, and even if we know it’s not right to stay up late, we do, ” Rogorga said. ” We need to understand the biological mechanisms of sleep deprivation that damage the body to find ways to prevent it. “

Source: Quanta Magazine

By Veronique Greenwood