The Good Kind of Brainwashing

About seven years ago, Maiken Nedergaard, a neuroscientist at New York State’s University of Rochester Medical Center (URMC), and colleagues noticed a striking change in the mice they were studying. The researchers were using a microscope to spy on the animals’ brains and track the movement of the cerebrospinal fluid (CSF) that surrounds and cushions that organ. When the mice were awake, only a trickle of fluid entered the brain. But whenever the animals were unconscious — either asleep or anesthetized — the floodgates opened. “We saw that CSF was recycled back into the brain in anesthetized and naturally sleeping mice, but this didn’t happen in awake mice,” says Nedergaard, an investigator with the Simons Collaboration on Plasticity and the Aging Brain (SCPAB).

The team had previously discovered a mechanism, which they dubbed the “glymphatic” system, that circulates CSF through the brain to rinse away metabolic wastes and other toxins. The new finding suggested the mice were ramping up the glymphatic system while they slept and in effect washing their brains. Nedergaard’s team has also found evidence the glymphatic system disposes of cellular trash. Together, Nedergaard says, these discoveries “opened up people’s eyes” to just how important glymphatic cleansing was in the brain.

Researchers now suspect that the glymphatic system is key for maintaining brain health, particularly as we get older. Circulation of fluid through the system appears to falter with age and in age-related illnesses. “The failure of that washing may underlie a great many diseases,” says Nedergaard’s former postdoctoral researcher, neuroscientist Jeff Iliff of the University of Washington in Seattle. Conversely, the system may maintain its performance in people who age well.

Nedergaard and other scientists are now trying to decipher precisely why and how the glymphatic system declines with age, and are studying ways to slow that decline — including exercise, better sleep and drugs that stimulate it.

The discovery of a hidden system

Given its crucial role in the brain, the glymphatic system remained obscure for a surprisingly long time. Nedergaard notes that as far back as the 1980s, researchers had seen hints of the system, such as signs of fluid movement within slices of brain tissue. But its physiological role wasn’t clear.

Her team first glimpsed the glymphatic system in action in the early 2010s. Iliff recalls sitting in a dark, frigid room at URMC and watching for a fluorescent green tracer molecule that had been injected into the base of a mouse’s skull. Through the eyepieces of the microscope, he saw a sheet of green creeping along one of the brain’s arteries. “It was almost like a halo,” he says. The discovery, which the scientists reported in 2012, indicated that CSF was infiltrating the brain via the circulatory system and then dispersing among its cells.

The finding solved a major riddle about the brain. Cells in the body continually pollute their surroundings with chemical wastes such as lactate, urea and broken-down proteins that need to be cleared away. In most tissues, lymphatic vessels collect this cellular sewage and deliver it to the bloodstream, allowing the liver and kidneys to detoxify or excrete it. Lymphatic vessels don’t enter the brain, however, and scientists had long wondered how that organ keeps clean.

Over the last eight years, aided by new imaging approaches for visualizing glymphatic flow in the living brain, researchers have assembled a portrait of the glymphatic system. Brain cells known as astrocytes are crucial for the system to operate. Resembling an octopus, these cells have multiple armlike projections tipped with pads called end-feet, which encircle the brain’s arteries and veins. Their loose grip leaves a gap called the perivascular space, through which CSF can flow. CSF follows arteries into the brain’s interior. There, the CSF leaves the perivascular space and spills into the surrounding brain tissue, mingling with neurons and other cells and sweeping up their chemical wastes and other noxious molecules. “It’s like the general garbage system,” says Nedergaard. “You don’t sort anything. It is going with the flow.”

Once the fluid reaches a nearby vein, it heads for the exit, entering the vein’s perivascular space and following it to the surface of the brain. The brain can also dispose of some wastes by transporting them across the blood-brain barrier into the bloodstream. How the blood-brain barrier interacts with the glymphatic system is still under investigation.

Scientists think that several sources power glymphatic circulation. Pulsations of brain arteries caused by the beating heart impel fluid through the perivascular spaces. Breathing may also draw the cerebrospinal fluid out of the brain and then drive it back in. Even posture contributes. Glymphatic flow is more vigorous when mice lie on their sides than when they lie on their backs or stomachs.

The glymphatic system’s slow decline

A growing body of evidence suggests that the glymphatic system slows with age and malfunctions in a variety of age-related diseases. Nedergaard’s team first made the connection to illness, showing that mice were slower to rid their brains of beta-amyloid, the protein that clumps in the brains of patients with Alzheimer’s disease, when their glymphatic system was impaired. Poor glymphatic circulation might also promote buildup of tau, the protein that amasses inside of cells in patients with Alzheimer’s disease. Researchers have also linked glymphatic deficiencies to Parkinson’s disease, amyotrophic lateral sclerosis, strokes, hypertension, diabetes and other diseases.

Nedergaard says that aging may take a toll on glymphatic function in several ways, thus setting people up for these illnesses. Blood vessels stiffen, dampening the pulsations that drive fluid through the perivascular spaces, and a weakening heart may provide less force to propel the CSF. As we get older, our brains shrink, and more cerebrospinal fluid may pool outside the organ. We produce less of the fluid with increasing age, which may slow its circulation.

The perivascular spaces also develop a drainage problem. Researchers think that one way the fluid departs from the spaces is by passing through a protein channel called aquaporin-4. Numerous copies of the protein stud the end-feet of astrocytes. Although some researchers still question aquaporin’s role, several independent labs have confirmed that the protein fosters glymphatic drainage. The number of aquaporin-4 molecules on the end-feet decreases in patients with Alzheimer’s disease and in older mice. The channels migrate to other parts of the astrocyte, where they can’t serve as outlets for the CSF.

Clogging in one of the glymphatic system’s exit chutes may also contribute to age-related decline. The system delivers “dirty” fluid to the boundary of the brain, where it is transferred to the body’s lymphatic system. Jonathan Kipnis, a neuroimmunologist at Washington University School of Medicine in St. Louis, and colleagues found that eliminating one of the system’s main outlets, known as meningeal lymphatic vessels, reduces the flow of CSF into the brain and the flow of contaminated fluid out. This CSF stagnation impairs cognitive function in mice — learning ability and memory decline. These findings suggest that the meningeal lymphatic vessels help the glymphatic system cleanse the brain and are necessary for normal cognition. Faulty meningeal lymphatic vessels may help explain why the glymphatic system malfunctions as we get older. Meningeal and glymphatic drainage is impaired with age, says Kipnis. In older animals and humans, the meningeal vessels narrow and become sparse.

Some mysteries about this drainage system still remain. For example, researchers still haven’t determined how fluid moves from the glymphatic system into the meningeal lymph vessels, because no direct connection between them has been identified.

Revitalizing the glymphatic system

Fortunately, it may be possible to slow the glymphatic system’s deterioration.

In a 2018 study, Kipnis and colleagues demonstrated that they could rejuvenate the system by bolstering the meningeal lymphatic vessels of old mice. The researchers used gene therapy to raise levels of VEGF-C, a growth factor that maintains lymphatic vessels and that becomes scarce with age. “If we provide VEGF-C, we can increase the coverage and function of vessels about to the level of young mice,” Kipnis says.

Boosting VEGF-C levels might also work in humans, although researchers still need to test the safety of this approach. Other approaches may also be feasible. Many of the steps that doctors routinely recommend for preserving our health as we age may also benefit the glymphatic system. For instance, because CSF circulation relies on arterial pulsations, maintaining cardiovascular health is important, says Nedergaaard. The best way of protecting the glymphatic system “is probably a lot of exercise,” she says.

Neurologist Daniel Claassen of Vanderbilt University Medical Center in Nashville, Tennessee, and colleagues are testing that idea in patients who have Parkinson’s disease, in which glymphatic circulation may be poor. The subjects will complete a noncontact boxing program that was developed for patients with the disease. To gauge glymphatic flow, he and his team will be tracking CSF fluid flow and beta-amyloid with PET and MRI scans. Although a hallmark of Parkinson’s disease is clumps of a different protein, alpha-synuclein, researchers don’t have techniques to visualize it in the living brain, Claassen says.

Iliff and his colleagues are trying a different approach to boost the glymphatic system: prodding it with a high-blood-pressure drug. His collaborators at the VA Puget Sound Health Care System and the University of Washington have launched a clinical trial with the drug prazosin, which they have found increases glymphatic flow in mice. “It’s been used for decades, so it’s generally safe,” Iliff says. “And it’s cheap.” The trial, which shut down temporarily because of the COVID-19 pandemic, will resume soon, he says. “The hope is that this will be a straightforward way to turn glymphatic function up.”

To study the glymphatic system in humans as they age, researchers need noninvasive ways to image it in the brain, Nedergaard says. Scientists can indirectly monitor glymphatic flow in humans by tracing the movement of gadolinium, a contrast agent commonly used for medical imaging, with MRI. The procedure involves injecting gadolinium into the space around the spinal cord, so for ethical reasons researchers have only performed these studies on patients who require the injections as part of their medical care.

Ian Harrison, a neuroscientist at University College London, and colleagues are evaluating diffusion tensor MRI, a technique that doesn’t require gadolinium injections. They have used this variety of MRI to observe CSF flow inside perivascular spaces in the brains of rats. The scientists are now validating their results, says Harrison, and they hope to begin testing the technique in humans.

Other imaging methods are also under development. “In three to four years, we will have four or five approaches that capture different facets of glymphatic function” in the human brain, says Iliff, although whether they will provide as much information as the gadolinium-based MRI scans is unclear.

A sleepy solution

Improving sleep may be another important avenue for enhancing the system’s functioning, because glymphatic activity cranks up as animals, including humans, slumber. Indeed, sleep may be the brain’s heavy-duty rinse cycle, according to findings from neuroscientist Laura Lewis of Boston University and colleagues. Her group used BOLD fMRI to measure blood oxygenation and CSF flow in the brains of awake and sleeping young people. When the subjects were awake, small, rapid pulses of CSF entered the brain about every 4 seconds. But when the subjects were asleep, Lewis and her colleagues discovered, large waves of CSF surged into the brain about every 20 seconds. Whether the CSF tide infiltrates the glymphatic system is still unclear, but the researchers assume it does.

The waves “may play a role in how sleep maintains brain health” by promoting fluid mixing, says Lewis, who is also an SCPAB investigator. She and her team determined that the CSF deluges occurred slightly after distinctive peaks of brain activity known as slow-delta waves, which the scientists detected with EEGs. Slow-delta waves typically occur when the brain is laying down long-term memories, suggesting the CSF surges are linked to this process.

Unfortunately, getting a good night’s sleep becomes harder and harder as we age. Not only do elderly people wake up more often during the night than do young people, but they also do not sleep as deeply. Most important, they don’t settle into the deepest level of sleep, known as NREM3, when glymphatic circulation crests. Young people, by contrast, spend most of the early part of the night in this sleep phase. Evidence suggests that sleep disruptions reduce brain cleansing. For example, one study found that the levels of beta-amyloid in the brain soared after one night of bad sleep. In addition, patients with age-related diseases like Parkinson’s and Alzheimer’s have abnormal sleep patterns. “Poor aging is linked to poor sleep,” says Nedergaard.

Her latest project, funded by the SCPAB, will investigate the link between sleep and glymphatic flow by studying two-year-old mice, about the equivalent of human septuagenarians. She and her colleagues will identify rodents that have maintained their sleep quality and memory, and then determine whether better glymphatic functioning is responsible. This work will be the first to test whether glymphatic efficiency has a role in healthy aging.

Meanwhile, Lewis and various collaborators also funded by the SCPAB are launching a study that will explore the impact of disrupted sleep on the glymphatic system and on cognitive function in humans. Using MRI, they plan to examine how impaired sleep in older people alters the flow of CSF, and to look for factors that predict when people are resistant to age-related decline in sleep quality.

The connection between sleep and glymphatic efficiency offers options for improving our health as we age, says Iliff. Although elderly people can’t slumber as deeply as 20-year-olds, they can improve their sleep habits — by going to bed at the same time every night, for instance — to give their glymphatic system the best chance of remaining healthy. The research also provides a warning to younger people who are stinting on sleep, says Iliff: “If you are a 45-year-old who sleeps five or six hours a night, you should probably start to think about how your brain will age over the next 30 years.”