New study reveals brain clearance of toxins is reduced during sleep and anesthesia

By Tarun Sai LomteReviewed by Lily Ramsey, LLMMay 15 2024

A recent study published in Nature Neuroscience reported that brain clearance is reduced during anesthesia and sleep.

Study: Brain clearance is reduced during sleep and anesthesia. Image Credit: Komsan Loonprom/Shutterstock.com

Background

Sleep represents a state of vulnerable inactivity. Given the risks of this vulnerability, it is assumed that sleep may confer some benefit. It has been suggested that sleep clears toxins and metabolites from the brain via the glymphatic system.

This notion has essential implications; for instance, diminished toxin clearance due to chronically poor sleep may aggravate Alzheimer’s disease.

How toxins and metabolites are cleared from the brain remains unclear, with disputes about the clearance mechanisms and anatomical pathways. According to the glymphatic hypothesis, the bulk fluid flow, driven by hydrostatic pressure gradients from arterial pulsations, actively clears brain parenchyma solutes during non-rapid eye movement sleep.

Further, at sedative doses, anesthetics enhance clearance. Whether sleep increases clearance by elevated bulk flow is unknown.

The study and findings

In the present study, researchers measured fluid movement and clearance in the brains of mice. First, the diffusion coefficient of fluorescein isothiocyanate (FITC)-dextran, a fluorescent dye, was determined. FITC-dextran was injected into the caudate putamen, and fluorescence was measured in the frontal cortex.

Initial experiments involved waiting for a steady state, bleaching the dye in a small tissue volume, and determining the diffusion coefficient from the unbleached dye's movement rate into the bleached zone.

The methodology was validated by measuring FITC-dextran diffusion in agarose brain phantom gels that were modified to approximate the brain's optical absorption and light scattering properties.

A hemispherical Gaussian distribution approximated the distribution of light. The recovery of fluorescence was recorded 30 seconds after bleaching.

The researchers noted that these (recording) data and the theoretically predicted time course were highly concordant. Further, the diffusion coefficient values were concordant with values estimated using a direct method (without photobleaching).

Next, the diffusion coefficient of FITC-dextran was measured in vivo, which was, once injected into the caudate putamen, detectable in the frontal cortex. Fluorescence peaked six to seven hours post-injection and waned at 6% per hour.

During the declining phase, the recovery from bleaching was recorded. The fluorescence recovery was consistent with theoretical predictions.

Effective tissue diffusion coefficient values were derived from the time courses, and the vigilance states (sleep, wake, and anesthesia) were determined. The average diffusion coefficient value was 32.1 μm²/s across all vigilance states, corresponding to a tortuosity of -2.5.

This was consistent with reported values for rodent neocortex and suggested that FITC-dextran movement within the cortex was mainly due to diffusion.

Of note, the diffusion kinetics were not different during anesthesia or sleep. Next, the team measured brain clearance during different vigilance states.

They used a small fluorescent dye, AF488, in mice injected with saline or anesthetic. This dye was reported to move freely in the parenchyma and could help quantify brain clearance accurately. Additionally, comparisons were made between wake and sleep states.

At the peak concentration, clearance was 70% to 80% in saline-injected mice, suggesting that standard clearance mechanisms were not disrupted. However, there was a substantial reduction in clearance with anesthetics (pentobarbital, dexmedetomidine, and ketamine-xylazine).

Further, clearance was also reduced in sleeping mice relative to awake mice. Nevertheless, the diffusion coefficient was not significantly different between anesthesia and sleep states.

Besides, electroencephalogram (EEG) power spectra were measured; this indicated a weak correlation between delta power and peak clearance, suggesting lower clearance with deeper sleep.

Histological experiments showed that the dye concentration three and five hours after injection was higher with ketamine-xylazine anesthesia and sleep. Data showed that AF488 redistribution was by diffusion alone and confirmed that both anesthesia and sleep inhibit clearance.

Conclusions

To conclude, the study illustrated reduced brain clearance during anesthesia and sleep, contradicting previous reports.

Clearance might vary across anatomical locations, but the extent of variation might be small. Nevertheless, clearance inhibition by ketamine-xylazine was significantly independent of the location.

Nicholas P. Franks, one of the authors, said – “The field has been so focused on the clearance idea as one of the key reasons why we sleep that we were very surprised to observe the opposite in our results.”

Notably, the findings are for a small dye with free movement in the extracellular space. As such, larger molecules may exhibit different behavior.

Besides, the exact mechanisms of how sleep and anesthesia impact brain clearance are unclear; however, the findings challenge the notion that the core function of sleep is brain toxin clearance.