31 May 2024

In recent years, several studies have suggested that the brain rinses itself of harmful waste, such as Aβ, during sleep. In the May 13 Nature Communications, researchers led by Nicholas Franks and William Wisden at Imperial College London cast doubt on those findings. They reported that instead of speeding clearance, sleep slowed the removal of a tracer dye from the brains of wild-type mice. They also found that neither sleeping, waking, nor anesthesia had any effect on how quickly dyes diffused throughout the brain. “Our results challenge the idea that the core function of sleep is to clear toxins from the brain,” the authors wrote.

Alan Verkman at the University of California, San Francisco, was impressed by the data. “I find the results convincing and important. They strongly refute the ‘glymphatic’ mechanism, which I and many other scientists do not believe,” he wrote to Alzforum. Others had concerns about methodology. “We found the results intriguing, but we believe the study has technical issues and potential for data misinterpretation,” wrote Benjamin Plog and Jonathan Kipnis, Washington University, St. Louis (comment below). A news piece in Science also noted that the methodology had drawn criticism.

Faster Cleanup While Awake? In the frontal cortices of mice anesthetized with ketamine-xylazine (top) or sleeping (middle), an injected dye clears more slowly than in awake mice (bottom). Quantification at right: anesthesia curve in red, sleep in blue, awake in black. [Courtesy of Miao et al., Nature Communications.]

The glymphatic hypothesis, first proposed by Maiken Nedergaard at the University of Rochester Medical School in New York 12 years ago, posits that astrocytes help drive the flow of cerebrospinal fluid through the brain parenchyma, washing away waste (Aug 2012 news). Subsequently, Nedergaard and others reported that this mechanism works best during sleep (Oct 2013 news; May 2014 conference news; Nov 2019 news). Not everyone was convinced, however, with some studies questioning the hypothesis in favor of clearance by diffusion (Asgari et al., 2016; Smith et al., 2017; Holter et al., 2017). 

To investigate further, joint first authors Andawei Miao and Tianyuan Luo measured the diffusion rate of molecules in wild-type mouse brain. They injected fluorescently labeled 4 kilodalton dextran into caudate putamen and allowed the dye to diffuse throughout the brain until it reached a steady state. Then they bleached the dye in a small region of the frontal cortex, and measured how quickly new dye moved into that region. This is an established way to determine diffusion rate in tissues (Thiagarajah et al., 2006). The dye dispersed at about 32 square meters per second, in good agreement with a previous report in rodent brain (Syková and Nicholson, 2008). Importantly, this rate did not change whether the mice were awake, sleeping, or anesthetized.

The authors next homed in on brain clearance, i.e., whether solutes are being removed from the brain as they circulate. For this experiment, they used the 0.5 kD dye AF488, which is small enough to move freely through parenchyma. Two to three hours after injection, the dye concentration in frontal cortices of awake mice was less than a third of what would be expected based on the diffusion rate, indicating that much of it had been cleared from the brain. In sleeping or anesthetized mice, on the other hand, about half the dye remained, implying that clearance ebbs during sleep and anesthesia. The authors confirmed the findings by sacrificing mice after injection and directly measuring the amount of dye in brain sections at set time points.

Why have so many previous studies reported higher clearance during sleep? The authors noted that most studies injected tracers into the CSF and measured how far into the cortex they penetrated, using that as a proxy for how fast solutes were moving out of the brain. However, because dye concentration in a brain region results from a balance between its influx and efflux rates, deeper penetration during sleep could be due to slower clearance, rather than faster movement, the authors suggested. “Under these circumstances, entry, exit, and redistribution of the marker are all occurring simultaneously, greatly confounding any quantification of clearance,” they wrote.

Jurgen Claassen, Radboud University Medical Center, Nijmegen, The Netherland, was not surprised by the findings. Ten years ago, he reported that as little as one night of sleep deprivation slowed clearance of Aβ from people’s brains (Ooms et al., 2014). But subsequent work from his lab seemed to disprove that idea. “These conflicting studies in mice and people show that we still do not know enough to say whether sleep has an important role to play in clearance,” he wrote to Alzforum (comment below).

Kaoru Yamada and Takeshi Iwatsubo, University of Tokyo, think clearance from the brain is more complicated than can be teased out with tracers. “Unlike the small dyes used in this study, many proteins present in the brain parenchyma are cleared not only by glymphatic clearance but also by a combination of mechanisms, including proteolytic degradation, phagocytosis by glial cells, and blood-brain barrier transport. Therefore, whether sleep and anesthesia have similar effects on other CNS proteins may warrant careful consideration,” they wrote to Alzforum. At the time of writing, Nedergaard had not responded to a request for comment.

If solute clearance does not speed up during sleep, then what explains the observed association between poor sleep and increased brain Aβ? One previous study suggested this was due to higher production of the peptide during a person’s wakeful hours, rather than to clearance during sleep (Jan 2018 news).

Claassen still thinks clearance plays a role. “Recent studies from different groups all clearly show that CSF flows dynamically, driven by hemodynamic and respiratory pressure oscillations, during wakefulness. I would still say it is likely that this CSF flow supports clearance beyond passive diffusion,” he wrote.—Madolyn Bowman Rogers and Tom Fagan

News Citations

1. Brain Drain—“Glymphatic” Pathway Clears Aβ, Requires Water Channel 16 Aug 2012
2. From ApoE to Zzz’s—Does Sleep Quality Affect Dementia Risk? 25 Oct 2013
3. Glymphatic Flow, Sleep, microRNA Are Frontiers in Alzheimer’s Research 6 May 2014
4. Deep Sleep Makes Waves for CSF 1 Nov 2019
5. Skimping on Sleep Makes For More Aβ in the Brain 15 Jan 2018

Paper Citations

1. Asgari M, de Zélicourt D, Kurtcuoglu V.
   Glymphatic solute transport does not require bulk flow.
   Sci Rep. 2016 Dec 8;6:38635.
   PubMed.
2. Smith AJ, Yao X, Dix JA, Jin BJ, Verkman AS.
   Test of the ‘glymphatic’ hypothesis demonstrates diffusive and aquaporin-4-independent solute transport in rodent brain parenchyma.
   Elife. 2017 Aug 21;6
   PubMed.
3. Holter KE, Kehlet B, Devor A, Sejnowski TJ, Dale AM, Omholt SW, Ottersen OP, Nagelhus EA, Mardal KA, Pettersen KH.
   Interstitial solute transport in 3D reconstructed neuropil occurs by diffusion rather than bulk flow.
   Proc Natl Acad Sci U S A. 2017 Sep 12;114(37):9894-9899. Epub 2017 Aug 28
   PubMed.
4. Thiagarajah JR, Kim JK, Magzoub M, Verkman AS.
   Slowed diffusion in tumors revealed by microfiberoptic epifluorescence photobleaching.
   Nat Methods. 2006 Apr;3(4):275-80.
   PubMed.
5. Syková E, Nicholson C.
   Diffusion in brain extracellular space.
   Physiol Rev. 2008 Oct;88(4):1277-340.
   PubMed.
6. Ooms S, Overeem S, Besse K, Rikkert MO, Verbeek M, Claassen JA.
   Effect of 1 night of total sleep deprivation on cerebrospinal fluid β-amyloid 42 in healthy middle-aged men: a randomized clinical trial.
   JAMA Neurol. 2014 Aug;71(8):971-7.
   PubMed.

External Citations

1. Science