CoChat
Sign Up
Cited by 173Cerebrospinal fluid and hydrocephalusSleep and Wakefulness ResearchTraumatic Brain Injury and Neurovascular Disturbances

Brain clearance is reduced during sleep and anesthesia

Open access
Miao, Andawei, Luo, Tianyuan, Hsieh, Bryan, Edge, Christopher J., Gridley, Morgan, Wong, Ryan Tak Chun, Constandinou, Timothy G., Wisden, William, Franks, Nicholas P.·2024·Nature Neuroscience
DOI 10.1038/s41593-024-01638-yPMC PMC11156584
Open this paper in CoChat — free account

Abstract

It has been suggested that the function of sleep is to actively clear metabolites and toxins from the brain. Enhanced clearance is also said to occur during anesthesia. Here, we measure clearance and movement of fluorescent molecules in the brains of male mice and show that movement is, in fact, independent of sleep and wake or anesthesia. Moreover, we show that brain clearance is markedly reduced, not increased, during sleep and anesthesia.

AI snapshot

Written from the paper’s full text.

TL;DR

In male mice, brain clearance of a small fluorescent dye was markedly reduced during sleep and anesthesia, not increased. Diffusion of a larger dye in cortex did not change with vigilance state, suggesting clearance changes are not due to altered diffusion.

Key findings

  • The diffusion coefficient of 4 kDa FITC-dextran in mouse frontal cortex did not change significantly with vigilance state or dexmedetomidine sedation (one-way ANOVA F(4,55) = 0.90; P = 0.47) or across the circadian cycle (F(5,64) = 0.88; P = 0.50). Mean D across all vigilance states was 32.1 ± 1.9 μm² s⁻¹ (n = 52), corresponding to tortuosity ~2.5.
  • Brain clearance of AF488 (~570 Da) was significantly reduced during sleep and anesthesia. Peak clearance was 70–80% in saline controls, but was significantly lower with dexmedetomidine (P = 0.0029), ketamine-xylazine (P = 0.0015), pentobarbital (P = 0.037), and sleep (P = 0.016).
  • Observed AF488 concentrations were significantly lower in the waking state than during dexmedetomidine, ketamine-xylazine, pentobarbital anesthesia, or sleep (all P < 10⁻⁶).
  • Histology confirmed higher dye concentrations during sleep and ketamine-xylazine anesthesia at both 3 h (sleep P = 0.0016; KET-XYL P < 10⁻⁶) and 5 h (sleep P < 10⁻⁴; KET-XYL P < 10⁻⁶). EEG showed a weak negative correlation between peak clearance and delta (0.5–4 Hz) power.

Methods

  • Adult male C57BL/6J mice (3–7 months) were implanted with a 200-μm optical fiber in frontal cortex and a guide cannula in caudate putamen. Diffusion was measured using in vivo photobleaching of 4 kDa FITC-dextran; clearance was measured by injecting AF488 into CPu and recording fluorescence in cortex over time.
  • Vigilance states were determined by EEG/EMG recording and automatic plus manual sleep scoring. Anesthesia experiments used dexmedetomidine (200 μg kg⁻¹ i.p.), ketamine-xylazine (100/20 mg kg⁻¹ i.p.), or pentobarbital (50 mg kg⁻¹ i.p.); sleep experiments used 5 h sleep deprivation followed by recovery sleep.
  • Data were fitted to diffusion/clearance equations and analyzed with ANOVA, paired t-tests, estimation statistics, and bootstrap confidence intervals.

Limitations

  • The paper states that it cannot rule out changes in pairwise flows in opposite directions over small distances, which might have averaged out, nor local fluid movement without bulk flow. It also notes that results are for a small dye that moves freely in extracellular space, and larger molecules may behave differently; the mechanism by which sleep and anesthesia inhibit clearance is unclear.
  • The paper states that data collection and analysis were generally not blind to experimental conditions, sample sizes were not predetermined, and some recordings/sections were excluded for poor fits or damage.
  • Evident from the text: experiments used only male mice and a single small tracer for clearance, so findings may not generalize to females, larger molecules, other brain regions, or humans.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1038/s41593-024-01638-y

Cites

36 of 36 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

18 of 24 cited sources checked · 6 supported · 0 flagged · accuracy not yet measured

  • Interpretationreasoning

    The mean diffusion coefficient corresponds to a tortuosity of ~2.5.

    “which corresponds, using equation (3), to a tortuosity of ~2.5 (having corrected the aqueous D to 37 °C using the Stokes–Einstein equation25).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [25] Sykova, E. & Nicholson, C. Diffusion in brain extracellular space. Physiol. Rev. 88, 1277–1340 (2008). source

      Section II.B defines tortuosity as λ = √(D/D*) and explains how to correct the free diffusion coefficient to the measurement temperature using the Stokes–Einstein equation. It supports the citing paper’s calculation method, but does not report the citing paper’s mean diffusion coefficient or establish that it yields a tortuosity of ~2.5. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • The authors state this as an assumption: The bleached fluorescent dye is distributed over a hemispherical volume with a Gaussian concentration profile.
    • The authors state this as an assumption: The volume recorded from is a hemispherical volume of radius R.
    • The authors state this as an assumption: The distance light penetrates to initiate bleaching is comparable to the distance light penetrates to record fluorescence, so R = σ.
  • Study design and conductown data

    The distribution of light intensity in agarose brain phantom gels was well approximated by a hemispherical Gaussian distribution.

    “We validated our methodology by measuring the diffusion of FITC–dextrans of various molecular weights in agarose ‘brain phantom’ gels, modified to approximate the light-scattering and optical-absorption properties of brain tissue22 and found (Extended Data Fig. 1) that the distribution of light intensity was well approximated by a hemispherical Gaussian distribution.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [22] Zhang, H. & Verkman, A. S. Microfiberoptic measurement of extracellular space volume in brain and tumor slices based on fluorescent dye partitioning. Biophys. J. 99, 1284–1291 (2010). source

      The Experimental validation section measures brain-tissue scattering and absorption and tests their effects on fluorescence, but does not describe a hemispherical Gaussian light-intensity distribution in agarose gels. Searches for that distribution and for optically modified agarose phantoms found no supporting passage. In the citing sentence, citation 22 appears attached to the brain-tissue optical properties; the Gaussian finding is presented as the citing authors’ own result. (LLM, conf=0.76)

      Experimental — accuracy not yet measured
  • Premisecitation

    Almost all experiments that have been interpreted as showing that sleep or anesthesia change brain clearance have involved introducing markers into the CSF, which then move into the brain parenchyma, confounding quantification of clearance.

    “Indeed, almost all the experiments that have been interpreted as showing that sleep or anesthesia change brain clearance have involved introducing markers into the CSF, which then move into the brain parenchyma14,26–30. Under these circumstances, entry, exit and redistribution of the marker are all occurring simultaneously, greatly confounding any quantification of clearance.”
    Quote in the paperSubstantial claim6 of 6 sources checked
    • cites [14] Hablitz, L. M. et al. Increased glymphatic influx is correlated with high EEG delta power and low heart rate in mice under anesthesia. Sci. Adv. 5, eaav5447 (2019). source
    • cites [26] Benveniste, H. et al. Anesthesia with dexmedetomidine and low-dose isoflurane increases solute transport via the glymphatic pathway in rat brain when compared with high-dose isoflurane. Anesthesiology 127, 976–988 (2017). source

      The Methods describe administering Gd-DTPA into CSF and measuring its uptake in brain parenchyma. The Discussion’s “Limitations of the study” says apparent clearance may reflect redistribution rather than true elimination, supporting the concern about quantification. This paper is an example of the method described, but it does not establish that almost all sleep- or anesthesia-related clearance experiments used it. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [27] Eide, P. K. et al. Sleep deprivation impairs molecular clearance from the human brain. Brain 144, 863–874 (2021). source

      The Discussion says this study added a tracer to CSF that subsequently entered the brain’s extravascular compartment, and identifies limitations of using that tracer to assess clearance. It does not establish that almost all sleep- or anesthesia-related clearance experiments used this approach, nor does it state that entry, exit, and redistribution simultaneously confound clearance quantification. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [28] Gakuba, C. et al. General anesthesia inhibits the activity of the glymphatic system. Theranostics 8, 710–722 (2018). source

      The Discussion (page unavailable) says this study and previous studies introduced tracers into the CSF and measured their influx into brain tissue. It also raises methodological concerns about tracer injection. It does not establish that almost all sleep- or anesthesia-related clearance experiments used this approach, or say that simultaneous entry, exit, and redistribution confound clearance measurements. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [29] Vinje, V. et al. Human brain solute transport quantified by glymphatic MRI-informed biophysics during sleep and sleep deprivation. Fluids Barriers CNS 20, 62 (2023). source

      The Methods (Data collection and approvals) describe a sleep-versus-sleep-deprivation study using a tracer injected into the CSF, and the Results (One-fourth of the tracers enter the brain) report its movement into brain tissue. The Discussion says CSF transport must be accounted for when quantifying brain tracer influx. The paper does not establish that almost all sleep or anesthesia experiments use this approach, or state that simultaneous entry, exit, and redistribution greatly confound clearance quantification. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [30] Ma, Q. et al. Rapid lymphatic efflux limits cerebrospinal fluid flow to the brain. Acta Neuropathol. 137, 151–165 (2019). source
  • Interpretationown data

    The movement of 4 kDa FITC-dextran in the cortex is predominantly by diffusion.

    “This is consistent with values reported for rodent neocortex25 and suggests that the movement of 4 kDa FITC-dextran in the cortex is predominantly by diffusion, a conclusion previously reached by others11,18,19.”
    Quote in the paperSubstantial claim3 of 4 sources checked
    • cites [25] Sykova, E. & Nicholson, C. Diffusion in brain extracellular space. Physiol. Rev. 88, 1277–1340 (2008). source
    • cites [11] Smith, A. J. et al. Test of the ‘glymphatic’ hypothesis demonstrates diffusive and aquaporin-4-independent solute transport in rodent brain parenchyma. eLife 6, e27679 (2017). source

      The Results section reports that dextran penetration into cortical parenchyma was consistent with diffusion, supporting the proposed mechanism. But it studied 10, 70, and 2000 kDa dextrans, not 4 kDa FITC-dextran, so it does not establish the claim for that specific tracer (page unavailable in our copy). (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites [18] Holter, K. E. et al. Interstitial solute transport in 3D reconstructed neuropil occurs by diffusion rather than bulk flow. Proc. Natl Acad. Sci. USA 114, 9894–9899 (2017). source
    • cites [19] Jin, B. J. et al. Spatial model of convective solute transport in brain extracellular space does not support a ‘glymphatic’ mechanism. J. Gen. Physiol. 148, 489–501 (2016). source

      The Discussion (page not provided) says tracer movement in gray matter is generally well described by simple diffusion and suggests that advection plays an insignificant role. The paper does not establish this specifically for 4 kDa FITC-dextran in the cortex, so it supports the conclusion at a broader level than the claim. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • The authors state this as an assumption: The bleached fluorescent dye is distributed over a hemispherical volume with a Gaussian concentration profile.
    • The authors state this as an assumption: The volume recorded from is a hemispherical volume of radius R.
    • The authors state this as an assumption: The distance light penetrates to initiate bleaching is comparable to the distance light penetrates to record fluorescence, so R = σ.
  • Findingown data

    Peak brain clearance was significantly reduced during DEX anesthesia.

    “For both anesthesia and sleep, the percentage of brain clearance was significantly reduced (two-tailed paired t-test): DEX (P = 0.0029), KET-XYL (P = 0.0015) or PENTO (P = 0.037) anesthesia or during sleep (P = 0.016).”
    Quote in the paperSubstantial claim
  • Classified as probably not a substantial claim. 32
  • May combine two claims. 10
  • The quote only partly states this claim. 1

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The mean D value is consistent with values reported for rodent neocortex.

    “This is consistent with values reported for rodent neocortex25”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [25] Sykova, E. & Nicholson, C. Diffusion in brain extracellular space. Physiol. Rev. 88, 1277–1340 (2008). source

      In IV.A, “Adult healthy brain” (page unavailable), the review reports diffusion measurements for rat and mouse neocortex, including tortuosity ranges, and distinguishes the effective diffusion coefficient (D*) from free diffusion (D). The citing sentence gives neither its mean D value nor the substance measured, so our copy establishes that rodent neocortical values are reported but cannot establish that the unspecified mean is consistent with them. (LLM, conf=0.76)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    The study's sample sizes are similar to those reported in previous publications3,4,12.

    “No statistical methods were used to predetermine sample sizes but our sample sizes are similar to those reported in previous publications3,4,12.”
    Quote in the paperNot a substantial claim3 of 3 sources checked
    • cites [3] Xie, L. et al. Sleep drives metabolite clearance from the adult brain. Science 342, 373–377 (2013). source

      The paper reports sample sizes of n = 6 and n = 10 mice in its experimental results (section labeled Abstract; page unavailable), so it is a previous publication that reports sample sizes. Our copy does not give the citing study’s sample sizes, however, and cannot establish that they are similar. (LLM, conf=0.75)

      Experimental — accuracy not yet measured
    • cites [4] Iliff, J. J. et al. A paravascular pathway facilitates CSF flow through the brain parenchyma and the clearance of interstitial solutes, including amyloid beta. Sci. Transl. Med. 4, 147ra111 (2012). source

      The Materials and Methods sections describe the animals and statistical methods, but give no sample sizes; the Statistics section directs readers to supplementary materials for details of each dataset. Searches for sample sizes and numbers of animals did not find figures in our copy that would allow comparison with the citing study. (LLM, conf=0.72)

      Experimental — accuracy not yet measured
    • cites [12] Mestre, H. et al. Flow of cerebrospinal fluid is driven by arterial pulsations and is reduced in hypertension. Nat. Commun. 9, 4878 (2018). source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    CSF outflow from the brain is markedly reduced by anesthetics.

    “Exactly how anesthetics and sleep inhibit brain clearance is unclear, although it is notable that CSF outflow from the brain is markedly reduced by anesthetics30.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [30] Ma, Q. et al. Rapid lymphatic efflux limits cerebrospinal fluid flow to the brain. Acta Neuropathol. 137, 151–165 (2019). source

      The Results (‘Outflow of CSF increases during awake conditions’; page unavailable) found significantly more CSF-infused tracer in the blood of awake mice than anesthetized mice, and the Discussion calls the difference substantial. This supports reduced CSF tracer outflow under anesthesia in mice, but the unqualified claim extends that finding beyond the studied population and tracer measure. (LLM, conf=0.82)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data

    The measured diffusion coefficients were in good agreement with literature values in aqueous solutions and their mass dependence.

    “Using this method, our measured diffusion coefficients were in good agreement with literature values in aqueous solutions23,24 and their mass dependence (inset to Fig. 1b).”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [23] Blassle, A. et al. Quantitative diffusion measurements using the open-source software PyFRAP. Nat. Commun. 9, 1582 (2018). source
    • cites [24] Pluen, A. et al. Diffusion of macromolecules in agarose gels: comparison of linear and globular configurations. Biophys. J. 77, 542–552 (1999). source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    The dye reached a peak in the frontal cortex about 6-7 h after injection.

    “At the start of the experiment, 4 kDa FITC-dextran was injected into the CPu (25 mg ml−1 in saline; 0.1 μl min−1 over 100 min), with injections being made (with different animals) throughout the 24-h cycle. The dye took about 2 h to be measurable in the frontal cortex, where it reached a peak about 6–7 h after injection (Extended Data Fig. 4a).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    For saline controls, peak concentrations were much lower than predicted by equation (2) but could be accounted for accurately by assuming clearance had occurred.

    “For the saline controls, the peak concentrations were much lower than that predicted by equation (2) but could be accounted for accurately by assuming clearance had occurred, as described by equations (8) and (9).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Thereafter, baseline fluorescence intensity in the frontal cortex declined slowly at about 6% per hour.

    “Thereafter, there was a slow decline in baseline intensity (~6% per hour), which was corrected for by fitting the baseline to a least-squares cubic spline curve.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    After injection of 4 kDa FITC-dextran into the CPu, the dye took about 2 h to become measurable in the frontal cortex.

    “At the start of the experiment, 4 kDa FITC-dextran was injected into the CPu (25 mg ml−1 in saline; 0.1 μl min−1 over 100 min), with injections being made (with different animals) throughout the 24-h cycle. The dye took about 2 h to be measurable in the frontal cortex, where it reached a peak about 6–7 h after injection (Extended Data Fig. 4a).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    The correction for the small spread of dye during the 30-s bleaching never exceeded 8%.

    “This small correction never exceeded 8% (Extended Data Fig. 1c).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In vivo, 4 kDa FITC-dextran injected into the CPu could be detected in the frontal cortex, where its fluorescence peaked at about 6-7 h postinjection, then slowly declined at ~6% per hour.

    “We then measured D in vivo using 4 kDa FITC-dextran, which after injection into the CPu, could be detected in the frontal cortex, where its fluorescence peaked at about 6–7 h postinjection, then slowly declined at ~6% per hour (Extended Data Fig. 4a).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    The average of the measured distances between the optical fiber and the cannula was 3.368 ± 0.064 mm (mean ± s.e.m.; n = 15).

    “The average of the measured distances was 3.368 ± 0.064 mm (mean ± s.e.m.; n = 15).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    For both the bleaching experiments and the clearance experiments, there were linear relationships between fluorescent intensity and dye concentration.

    “For both the bleaching experiments and clearance experiments, there were linear relationships between fluorescent intensity and dye concentration.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The spread of AF488 in histology was Gaussian, with characteristic widths roughly in line with those predicted using diffusion coefficients derived from photometry.

    “As expected, (equation (8)), the spread was Gaussian (fitted curves in Fig. 3b), with characteristic widths roughly in line with those predicted using the diffusion coefficients derived from the photometry experiments.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductcitation

    There was excellent agreement between the photometry data and equation (8), with discrepancies at small times possibly due to dye finding its way across the brain via the ventricles.

    “There was excellent agreement between the photometry data and equation (8), with the discrepancies at small times possibly being due to dye finding its way across the brain via the ventricles16.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [16] Pla, V. et al. A real-time in vivo clearance assay for quantification of glymphatic efflux. Cell Rep. 40, 111320 (2022). source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    For the histology experiments, brain sections that were substantially damaged were excluded from the quantitative analysis.

    “For the histology experiments, brain sections that were substantially damaged were excluded from the quantitative analysis.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    For the diffusion coefficient measurements, bleaching recordings that could not be fitted by the custom curve-fitting algorithm were excluded.

    “For the diffusion coefficient measurements, bleaching recordings that could not be fitted by the custom curve-fitting algorithm were excluded.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    No statistical methods were used to predetermine sample sizes.

    “No statistical methods were used to predetermine sample sizes but our sample sizes are similar to those reported in previous publications3,4,12.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Normality was confirmed using the Kolmogorov-Smirnov test.

    “Normality was confirmed using the Kolmogorov–Smirnov test.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisereasoning

    For values of x that are small compared to L (~20% or less), C(x,t) is very insensitive to x because of the cosine term.

    “Because of the cosine term, for values of x that are small compared to L (~20% or less), C(x,t) is very insensitive to x.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Data collection and analysis were generally not performed blind to the conditions of the experiments.

    “Data collection and analysis were generally not performed blind to the conditions of the experiments.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisereasoning

    If clearance occurs, the height of the peak would be reduced.

    “If clearance occurs, the height of the peak would be reduced (Fig. 2c and equation (8)).”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Anesthetics at sedative doses induce states resembling deep NREM sleep.

    “Anesthetics at sedative doses, which induce states resembling deep NREM sleep2,13, were also reported to increase clearance3,14,15.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [2] Franks, N. P. & Wisden, W. The inescapable drive to sleep: overlapping mechanisms of sleep and sedation. Science 374, 556–559 (2021). source
    • cites [13] Franks, N. P. General anaesthesia: from molecular targets to neuronal pathways of sleep and arousal. Nat. Rev. Neurosci. 9, 370–386 (2008). source

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductown data

    The results cannot rule out changes in pairwise flows in opposite directions over small distances in the surrounding tissue, which might have averaged out, so brain clearance might nonetheless have changed.

    “but our results cannot rule out changes in pairwise flows in opposite directions over small distances in the surrounding tissue, which might have averaged out, so that brain clearance might, nonetheless, have changed.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S4 (based).

    Premisenone given

    The concentration of dye in any brain region is the difference between its rate of arrival and its rate of departure.

    “However, the concentration of dye in any brain region will always be the difference between its rate of arrival and its rate of departure”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Study design and conductnone given

    The spread might be enhanced by local fluid movement without bulk flow; however, these do not change with vigilance state.

    “We cannot rule out that spread might be enhanced by local fluid movement without bulk flow; however, these do not change with vigilance state.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    After automatic scoring, each vigilance state was screened and confirmed manually.

    “Nonetheless, after automatic scoring, each vigilance state was then screened and confirmed manually afterwards.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning

    If the photometry diffusion coefficients reflect pure diffusion, they would correspond to a tortuosity of ~1.4.

    “If these diffusion coefficients reflect pure diffusion, then they would correspond to a tortuosity of ~1.4.”
    Quote in the paperNot a substantial claim
    • The authors state this as an assumption: The bleached fluorescent dye is distributed over a hemispherical volume with a Gaussian concentration profile.
    • The authors state this as an assumption: The volume recorded from is a hemispherical volume of radius R.
    • The authors state this as an assumption: The distance light penetrates to initiate bleaching is comparable to the distance light penetrates to record fluorescence, so R = σ.

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    In the anesthesia experiments, mice were injected with either an anesthetic or saline, 1 week apart and in random order.

    “For the anesthesia experiments, mice were injected with either an anesthetic (see ‘Anesthesia’) or saline, 1 week apart and in random order.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    For the photometry recordings, poor fits to the theoretical curves were excluded and recordings where one of the paired recordings (saline or anesthetic; or sleep and wake) was not successful were excluded.

    “For the photometry recordings, poor fits to the theoretical curves were excluded and recordings where one of the paired recordings (saline or anesthetic; or sleep and wake) was not successful.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductown data

    There was excellent agreement between the fluorescence recovery data and the time course predicted using equations (4) and (5).

    “There was excellent agreement between these data and the time course predicted using equations (4) and (5) (Methods and Extended Data Fig. 2).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    In the sleep experiments, wake and recovery sleep recordings were made on the same animal, 1 week apart, in random order.

    “For the sleep experiments, mice were sleep deprived for 5 h and then allowed to sleep (Extended Data Fig. 7). Recordings were made either during the wake period (for 5 h) or during the recovery sleep period, starting at the first sleep episode. These recordings were made on the same animal, 1 week apart and again in random order.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    The spread of light in a brain slice confirmed that the distribution was well approximated by a hemispherical Gaussian distribution.

    “The spread of light in a brain using a brain slice (Methods) confirmed that the distribution was also well approximated by a hemispherical Gaussian distribution (Extended Data Fig. 4b).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    A difference of ~35% in D would have been detected.

    “(A difference of ~35% in D would have been detected.)”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    The diffusion coefficients agreed well with values obtained using a direct method that did not involve photobleaching.

    “Our diffusion coefficients also agreed well (Fig. 1c) with values obtained using a direct method (Extended Data Fig. 3) that did not involve photobleaching.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    The automatic sleep-scoring algorithm was done blind.

    “However, the automatic sleep-scoring algorithm was done blind and the vigilance states then checked manually.”
    Quote in the paperNot a substantial claim

  • The quote only partly states this claim.

    Study design and conductown data

    The automatic sleep-scoring algorithm had high sensitivity (0.91-0.97) and specificity (0.92-0.99) for Wake, NREM and REM compared with experienced human sleep scorers.

    “The sensitivity and specificity when compared to experienced human sleep scorers were very high (see below).”
    • Also stated in: § EEG/EMG recording and sleep scoring · plain
      “| Sensitivity | 0.91 | 0.97 | 0.91 | 0.94 | 0.93 | 0.95 |”
    • Also stated in: § EEG/EMG recording and sleep scoring · plain
      “| Specificity | 0.98 | 0.92 | 0.99 | 0.96 | 0.96 | 0.98 |”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    The fluorescence recovery in vivo agreed well with theoretical predictions.

    “As with the gel experiments described above, the fluorescence recovery agreed well with the theoretical predictions (Extended Data Fig. 4c) and we derived values for the effective tissue D from the time courses, while also determining the vigilance states (Extended Data Fig. 4d).”
    Quote in the paperNot a substantial claim
37 of 37

  • The authors state this as an assumption.

    The bleached fluorescent dye is distributed over a hemispherical volume with a Gaussian concentration profile.

    “We assume that, following bright illumination, the bleached fluorescent dye is distributed over a hemispherical volume with a concentration, Q(s), that falls off as a Gaussian distribution (see main text and Extended Data Figs. 1b and 4b for experimental confirmation):1 Q(s)=Q(0)exp(−s²/2σ²)(s≥0)”

  • The authors state this as an assumption.

    The volume recorded from is a hemispherical volume of radius R.

    “If we assume the volume being recorded from is a hemispherical volume of radius R”

  • The authors state this as an assumption.

    The distance light penetrates to initiate bleaching is comparable to the distance light penetrates to record fluorescence, so R = σ.

    “If we assume that the distance that light penetrates into the tissue to initiate bleaching will be comparable to the distance light penetrates to record the fluorescence as dye diffuses back into the bleached volume, then we can set R = σ.”

  • The authors state this as an assumption.

    In the clearance experiments, the dye spread according to equation (8), with σ as the characteristic width of the initial Gaussian distribution of dye.

    “We assumed that the dye spread according to equation (8) (see Fig. 2 for experimental verification and also Extended Data Fig. 6) but where σ is now the characteristic width of the initial Gaussian distribution of dye, rather than the width of the bleached dye, as was the case for the bleaching experiments.”

  • The authors state this as an assumption.

    The loss of dye due to brain clearance can be modeled by multiplying the diffusion equation by (1−t/(t+τ)), where τ is the half time for clearance.

    “To account for the loss of dye due to brain clearance, the equation was multiplied by a term (1−t/(t+τ)), where τ is the half time for clearance, giving:”
5 of 5

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Miao et al. give no justification for the mathematical form of Eq. 8.

    “Miao et al.7 give no justification for the mathematical form of Eq. 8”

    From A curious concept of CNS clearance (2025) · cites this paper as Miao et al. [7]

    The Methods section, “Protocol for measuring brain clearance” (page unavailable), gives a rationale for Eq. 8: it uses the dye-spread equation, cites experimental verification, and adds a factor to account for dye loss through clearance. It does not explain why that particular clearance factor was chosen, but the unqualified claim that it gives no justification for Eq. 8’s form is contrary to the passage. (LLM, conf=0.78)

    Checked by a reasoning model — may be wrong.

    Experimental — accuracy not yet measured

  • Miao et al. estimate tracer diffusivity by fitting experimental measurements to Eq. 8.

    “Miao et al.7 estimate the tracer diffusivity by fitting experimental measurements to Eq. 8”

    From A curious concept of CNS clearance (2025) · cites this paper as Miao et al. [7]

    The Fig. 1b caption in the Main section says the photobleaching measurements were fitted to equation (5) to obtain the diffusion coefficient D. The Methods section on measuring brain clearance instead uses equation (8) to model dye concentration when clearance is present. The paper therefore assigns the claimed diffusivity fit to a different equation. (LLM, conf=0.90)

    Checked by a reasoning model — may be wrong.

    Experimental — accuracy not yet measured

  • Miao et al. ignore the possibility that diffusivity and advection may change between wake and anesthesia/sleep.

    “ignoring the possibility that diffusivity and advection may change between wake and anesthesia/sleep”

    From A curious concept of CNS clearance (2025) · cites this paper as Miao et al. [7]

    The Main section (discussion of Fig. 1d,e and Extended Data Fig. 5; page not provided) explicitly tests whether diffusion changes across vigilance states and says the results cannot rule out changes in small-scale flows. The paper therefore considers, rather than ignores, the possibility of changes in diffusivity and fluid movement. (LLM, conf=0.90)

    Checked by a reasoning model — may be wrong.

    Experimental — accuracy not yet measured

  • The data of Miao et al. fails to support meaningful conclusions, whether regarding glymphatic clearance or other aspects of brain fluid transport.

    “Consequently, the data fails to support meaningful conclusions, whether regarding glymphatic clearance or other aspects of brain fluid transport.”

    From A curious concept of CNS clearance (2025) · cites this paper as Miao et al. [7]

    The Main section says the experiments show reduced brain clearance during sleep and anesthesia and reports no significant change in diffusion kinetics. It acknowledges limits on what its measurements can rule out, but draws substantive conclusions rather than saying its data cannot support meaningful conclusions about brain transport. Page numbers are unavailable in our copy. (LLM, conf=0.90)

    Checked by a reasoning model — may be wrong.

    Experimental — accuracy not yet measured

  • Miao et al.'s design is fundamentally uninformative.

    “Its design is fundamentally uninformative”

    From A curious concept of CNS clearance (2025) · cites this paper as Miao et al. [7]

    The Main section says the dye-based approach can accurately quantify brain clearance and reports results from it. That is contrary to the attributed claim that the study’s design is fundamentally uninformative. Page numbers were not available in our copy. (LLM, conf=0.85)

    Checked by a reasoning model — may be wrong.

    Experimental — accuracy not yet measured

Cites

Flagged first, then by citations

Cited by

Sorted by citations

Related

Authors
Miao, A., Luo, T., Hsieh, B., Edge, C. J., Gridley, M., Wong, R. T. C., Constandinou, T. G., Wisden, W., & Franks, N. P.
Affiliations
  • UK Dementia Research Institute (GB)
  • Zunyi Medical University (CN)
  • Imperial College London (GB)
Corresponding author
  • Wisden, William
  • Franks, Nicholas P.
ORCID
Venue
Nature Neuroscience
Publisher
Springer Science and Business Media LLC
Published
2024-05-13
Volume
27
Issue
6
Pages
1046-1050
ISSN
  • 1097-6256 (ISSN-L)
  • 1546-1726
DOI
10.1038/s41593-024-01638-y
PMC
PMC11156584
OpenAlex
W4396862450
Semantic Scholar
0a334f797fcc8b5a87a04a2aba4c55daec7987d2
Cited by
172
References
36
Citations by year
2026: 59 · 2025: 91 · 2024: 22
FWCI
43.09
Type
journal article
Language
en
Update notices
correction (2024-06-14)
Open access
Licence
Locations
  • Nature Neuroscience (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
  • Wellcome Trust (220759/Z/20/Z, 104931/Z/14/Z)
  • RCUK | Biotechnology and Biological Sciences Research Council (BB/L105129/1)
  • UK Dementia Research Institute
Topics
  • Health Sciences › Medicine › Clinical Neurology › Cerebrospinal fluid and hydrocephalus
  • Life Sciences › Neuroscience › Cognitive Neuroscience › Sleep and Wakefulness Research
  • Health Sciences › Medicine › Clinical Neurology › Traumatic Brain Injury and Neurovascular Disturbances
Keywords
brain clearance, sleep, anesthesia, brain waste clearance, brain metabolites, sleep function, mice
MeSH terms
Anesthesia, Animals, Brain, Male, Mice, Inbred C57BL, Sleep, Wakefulness, Mice

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-11-07.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-11-07.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review