Kelly, Kathleen M.; Mikell, Charles B.; McKhann, Guy M. II
“Sleep that knits up the ravell'd sleave of care,
The death of each day's life, sore lab; our's bath, Balm of hurt minds…”
One of the remaining great mysteries of biology is the purpose of sleep. All species require sleep in some capacity, and the detrimental effects from lack of sleep have been shown countless times. Yet despite extensive research, the precise role of this vital process remains elusive. A recent study from the Nedergaard lab at the University of Rochester suggests that sleep is, in fact, a homeostatic mechanism for the brain that promotes clearance of neurotoxins.1 Xie et al hypothesize that sleep is characterized by changes in the glymphatic system (a term used to refer to the convection-like process by which the brain exchanges interstitial fluid [IF] for cerebrospinal fluid [CSF]). They posit that deep sleep increases the volume of the interstitial space, thereby increasing convective exchange between IF and CSF as compared to the awake state. This mechanism could be important for clearance of dangerous neurotoxins, such as beta-amyloid (Aβ).
To test this hypothesis, the team initially compared CSF influx of sleeping, anesthetized, and awake mice. Fluorescent radiotracers were infused into the cisterna magna, and influx was measured in real time using 2-photon imaging. Electrocorticography (ECoG) and electromyography (EMG) were recorded to monitor brain activity. In the first experiment, fluorescein isothiocyanate (FITC)-dextran (3kD, green) was infused during a time when mice typically sleep (12-2 pm), with a subsequent infusion of Texas red dextran (3kD, red) after the animal had been awakened. Unexpectedly, periarterial and parenchymal influx of CSF was reduced by 95% when the mice were awake (Figure). The experiment was then repeated with a new cohort during a time when mice are typically awake (8-10 pm) to determine whether brain activity altered CSF influx. After the initial infusion of FITC-dextran, the mice were anesthetized with ketamine/xylazine and infused with Texas red dextran 15 minutes later. Anesthesia significantly increased influx of CSF tracer to the level of sleeping mice.
Figure. Wakefulness ...Image Tools
The authors then sought to address the mechanism of these changes in CSF dynamics using an established technique for quantifying changes in interstitial volume. They evaluated the volume and tortuosity of the interstitial space in real-time by initiating iontophoresis of tetramethylammonium (TMA+) with a microelectrode in head-fixed mice while continuously recording with a TMA+ sensitive electrode approximately 150 microns away. A smaller interstitial space would cause less dilution of the TMA+, resulting in a higher detected signal. Similar to the first set of experiments, a cohort of mice was tested during a normal time for sleep and then awakened, and a separate cohort of mice was tested during a normal time for activity and then anesthetized. While tortuosity of the interstitial space did not change between brain states, the volume fraction was significantly larger in the sleeping and anesthetized states (22-24%) than in the awake state (13-15%, P < .01). The decrease in interstitial space in the awake state likely causes an increased tissue resistance to interstitial fluid flux and CSF inflow.
As Aβ has been shown to be cleared by the glymphatic system,2 the third experiment evaluated differences in clearance of this metabolite between brain states. Radiolabeled 125Aβ1-40 was injected intracortically into sleeping, anesthetized, and awake mice, and the brains were harvested 10 to 240 minutes afterwards. There was significantly decreased clearance of Aβ in the awake state compared to sleeping and anesthetized states, and no difference in clearance between sleeping and anesthetized states. Since Aβ clearance is partially mediated by receptor transport across the blood-brain barrier, the experiment was repeated with 14C-inulin, an inert tracer. Similar increases in clearance were noted during sleep and anesthetized states, thereby indicating that the majority of both substances were cleared by a common mechanism, likely the glymphatic system.
In the final experiment, the authors sought to identify the modulator of glymphatic influx in the sleep-wake state. Since noradrenergic signaling has been implicated in arousal3,4 as well as changes in cell volume in peripheral tissues,5 they hypothesized that increased norepinephrine in the awake state causes an increase in cell volume, thereby decreasing interstitial volume. To test this hypothesis, CSF influx was measured using the same method as in the first experiment, only this time the mice received either a cocktail of adrenergic receptor antagonists (prazosin, atipamezole, and propranolol) or vehicle 15 minutes prior to fluorescent tracers. The adrenergic antagonist group demonstrated an increase in CSF influx to the level of sleeping or anesthetized mice. Subsequently, interstitial volume was measured after infusion using the same TMA+ ionophoretic method as described above. Recordings showed an increase of interstitial volume from 14.3% to 22.6%, similar to previous recordings in the awake vs the sleeping or anesthetized states, respectively. After adrenergic antagonist infusion, ECoG confirmed an increase in slow-wave activity consistent with a sleep-like state.
This study presents a compelling case that the state of arousal modulates interstitial volume, which in turn modulates CSF influx and convective exchange of metabolites. The universal “need for sleep” may represent a homeostatic demand on the organism to clear toxic metabolites. These data should be viewed in light of recent literature suggesting that the adverse consequences of sleep deprivation (common in neurosurgical training and practice) are serious. Though several studies have showed relatively minor or non-existent decrements in surgical performance following sleep deprivation6,7 the long-term harm may be to the surgeon. Especially concerning are recent findings linking poor sleep in adults to accumulation of Aβ.8 Further research will be needed to understand how the effects of sleep deprivation might be mitigated. These studies will be of interest to the entire neurosurgical community.
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