New method reignites controversy over brain clearance during sleep

New method reignites controversy over brain clearance during sleep

Research results published last week showed that injecting a tracer directly into the brain tissue of mice, rather than into the cerebrospinal fluid, slowed brain clearance during sleep and under anesthesia, supporting the glymphatic system. They have concerns about this technology.

New research suggests that the brain excretes less waste during sleep and under anesthesia than in other conditions. This directly contradicts previous findings suggesting that sleep triggers this process. This discovery has sparked new debate on social media and elsewhere about the glymphatic system hypothesis, in which toxins are removed from the brain during sleep through convection of cerebrospinal fluid.

His new study, published May 13 in Nature Neuroscience, suggests that fluid diffusion is involved in the transport of waste products throughout the brain. Jason Rihel, a professor of behavioral genetics at University College London, used a different method than previous studies, injecting the tracer into the mice's brain tissue rather than into the cerebrospinal fluid. say this is likely a more reliable way to understand how fluid moves through densely packed neurons. He was not involved in the brain clearance study.

The findings have led some sleep researchers, including Richel, to question the existence of the glymphatic system and whether brain clearance is related to sleep-wake states, he says. . But leading proponents of the sleep-induced clearance theory have pushed back against the study's methods. Maiken Nedergaard, a professor of neurology at the University of Rochester Medical Center, said the new study is "misleading" and "woefully inadequate," and that her 2013 study of brain clearance led to the glymphatic system hypothesis. She plans to challenge the study in a proposed "Matters Arising" commentary in Nature Neuroscience.

Inserting a needle into the brain, as the team behind the new study did, damages tissue, and injecting fluid increases intracranial pressure, says Jonathan Kipnis, a professor of pathology and immunology at Washington University School of Medicine in St. Louis. Kipnis and his colleagues published a study in February supporting the glymphatic system hypothesis, suggesting that neuronal activity drives brain clearance.

"When you inject it into the brain, it confuses the system," Kipnis says. "That's why we always injected into the cerebrospinal fluid."

Nick Franks, professor of biophysics and anesthesiology at Imperial College London and principal investigator on the new study, said glymphatic theory was initially a "very poor" explanation of why we need sleep. "It seemed like a good idea," he said. Previous glymphatic studies have measured waste removal during sleep and other states by introducing markers into an animal's cerebrospinal fluid and tracking how quickly the markers enter the brain. Ta. A 2013 study found that increased fluid flow to the brain reflects an increased volume of interstitial space, allowing for more efficient waste removal through convective fluid flow. .

But Franks wanted to measure fluid movement in the brain more directly, "without using that logic," he says. He and his colleagues injected mice with a fluorescent dye into the caudate-putamen brain region through an implanted cannula. Optical fibers implanted approximately 3 millimeters apart in the frontal cortex tracked the concentration of fluorescent markers as the mice entered various states of wakefulness or sedation during a 12-hour light or dark period.

Bright light delivered from an optical fiber to the caudate putamen photobleachs the pigment there, and the researchers measure how quickly the unbleached pigment re-enters the area within 24 hours. It is now possible to calculate the "diffusion coefficient" in the brain. And this diffusion coefficient matched those measured in agarose gels and mouse brain sections in other experiments.

Diffusion coefficients were the same when the animals were awake, in non-REM and REM sleep, in light and dark conditions, and under dexmedetomidine sedation. This suggests that the dye spread through cortical migration rather than through convective changes. The glymphatic hypothesis is predictive, Franks says.

Franks and his team also found that 12 hours after giving anesthesia or saline to mice, or 5 hours or 12 hours of wakefulness during wakefulness, compared to those used in previous studies on glymphatic clearance. The concentration of tiny dyes, even hundreds of Daltons smaller, was measured. sleep. The concentration of dye reaching the optical fiber was lower after saline injection or in awake animals than under anesthesia or during sleep. Also, mouse brain slices showed more pigment when taken during sleep or under anesthesia.

In summary, the results show that the dye diffuses into the brain regardless of the animal's condition, and that the brain secretes more dye when awake or after saline ingestion than during sleep or anesthesia. says Professor Franks.

"Relatively speaking, more [dye] was retained during anesthesia and during sleep...and that was exactly the opposite of what the glymphatic hypothesis predicted," Franks says.

Many techniques are being misused in the new research, Nedergaard said, and she plans to elaborate on her criticisms in a contribution to Nature Neuroscience. For example, direct injections into the brain require more controls than were used by Franks et al. to examine the glial scar and ensure that the amount of injected dye actually reaches the tissue. animals are needed. The cannula should have been clamped for 30 minutes after fluid injection to ensure there was no reflux, she added. Animals in the sleep group were a model of sleep recovery from 5 hours of sleep deprivation, rather than natural sleep. This is what makes her different, she explains, adding, "It's misleading."

"They don't realize that there are many fundamental flaws in the experimental setup," she says.

More broadly, Nedergaard says, measurements in the brain cannot demonstrate brain cleansing. “The idea is that if you take the trash can from the kitchen to the garage, it won’t get clean,” she said.

According to Nedergaard, there is no glymphatic pathway that transports fluid from the injection site deep in the brain to the frontal cortex, where optical measurements are taken. She added that the two regions are likely separated by white matter tracts. "Why should waste be sent in this direction?"

Technical issues such as intracranial pressure and injection methods are legitimate concerns that make it difficult to evaluate new experiments, Richel said. However, mathematical modeling of diffusion and careful consideration of diffusion techniques within gels are compelling. The results are also consistent with previous theoretical models that suggest diffusion rather than convection, he explains. "And if you don't have that kind of convection, you don't need a glymphatic system, at least not as designed."

Different molecules may be cleared from the brain at different rates or under different conditions, Richel said. Previous glymphatic studies used biologically relevant molecules such as beta-amyloid, he says, while the new study used an inert dye.

The new study seems to contradict the glymphatic study, but it could just be a different interpretation of the same results, Franks says. The fact that the markers injected into the cerebrospinal fluid in the previous study penetrated deep into the cerebral cortex during sleep and under anesthesia "could equally well be explained by reduced clearance," he says.

The glymphatic model is more than 10 years old, Nedergaard says, adding: "You always simplify the model. We're perfectly happy to revise the model. But to argue that the glymphatic hypothesis is wrong, you have to repeat the original method and show why it's wrong," she says.

The idea of ​​a flipped interpretation "just turns everything upside down," says Ali Amidi, an associate professor of psychology and behavioral sciences at Aarhus University, who was not involved in the study. "And it's not often that you see such a difference in interpretation of the same result."

Professor Kipnis said better tools are needed to reconcile the differences in interpretation between sleep-induced studies and this study, particularly genetically engineered mouse models that can direct tracers to specific brain areas at specific times. Says. "And I think anyone who thinks we've solved the sleep problem or we've solved the brain cleansing problem is very naive," he says.

Franks said he expected a backlash to the study, which is one reason he didn't present it at the conference, to avoid getting caught up in the "zeitgeist."

"I don't want people to tell me I'm wrong," he says. ``Get out of there.'' Publish your essay, hope for the best, and then see what happens. ”

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