Chronic sleep fragmentation impairs brain interstitial clearance in young wildtype mice.

Accumulating evidence shows that most chronic neurological diseases have a link with sleep disturbances, and that patients with chronically poor sleep undergo an accelerated cognitive decline. Indeed, a single-night of sleep deprivation may increase metabolic waste levels in cerebrospinal fluid. However, it remains unknown how chronic sleep disturbances in isolation from an underlying neurological disease may affect the glymphatic system. Clearance of brain interstitial waste by the glymphatic system occurs primarily during sleep, driven by multiple oscillators including arterial pulsatility, and vasomotion. Herein, we induced sleep fragmentation in young wildtype mice and assessed the effects on glymphatic activity and cognitive functions. Chronic sleep fragmentation reduced glymphatic function and impaired cognitive functions in healthy mice. A mechanistic analysis showed that the chronic sleep fragmentation suppressed slow vasomotion, without altering cardiac-driven pulsations. Taken together, results of this study document that chronic sleep fragmentation suppresses brain metabolite clearance and impairs cognition, even in the absence of disease.

Association of intra-shift nap duration with heart rate variability in medical night shift workers.

Napping during night shifts effectively reduces disease risk and improves work performance, but few studies have investigated the association between napping and physiological changes, particularly in off-duty daily lives. Changes in the autonomic nervous system precede diseases like cardiovascular disease, diabetes, and obesity. Heart rate variability is a good indicator of autonomic nervous system. This study aimed to investigate the link between night shift nap durations and heart rate variability indices in the daily lives of medical workers. As indicators of chronic and long-term alterations, the circadian patterns of heart rate variability indices were evaluated. We recruited 146 medical workers with regular night shifts and divided them into four groups based on their self-reported nap durations. Heart rate variability circadian parameters (midline-estimating statistic of rhythm, amplitude, and acrophase) were obtained by obtaining 24-h electrocardiogram on a day without night shifts, plotting the data of the heart rate variability indices as a function of time, and fitting them into periodic cosine curves. Using clinical scales, depression, anxiety, stress, fatigue, and sleepiness were assessed. Linear regression analysis revealed a positive relationship between 61-120-min naps and 24-h, daytime, and night-time heart rate variability indices, and the parasympathetic activity oscillation amplitude (indexed by high-frequency power, the square root of the mean of the sum of squares of differences between adjacent normal intervals, standard deviation of short-term R-R-interval variability) within one circadian cycle. This study indicated that napping for 61-120 min during night shifts could benefit medical workers' health, providing physiological evidence to promote nap management.

Astrocytic chloride is brain state dependent and modulates inhibitory neurotransmission in mice.

Information transfer within neuronal circuits depends on the balance and recurrent activity of excitatory and inhibitory neurotransmission. Chloride (Cl-) is the major central nervous system (CNS) anion mediating inhibitory neurotransmission. Astrocytes are key homoeostatic glial cells populating the CNS, although the role of these cells in regulating excitatory-inhibitory balance remains unexplored. Here we show that astrocytes act as a dynamic Cl- reservoir regulating Cl- homoeostasis in the CNS. We found that intracellular chloride concentration ([Cl-]i) in astrocytes is high and stable during sleep. In awake mice astrocytic [Cl-]i is lower and exhibits large fluctuation in response to both sensory input and motor activity. Optogenetic manipulation of astrocytic [Cl-]i directly modulates neuronal activity during locomotion or whisker stimulation. Astrocytes thus serve as a dynamic source of extracellular Cl- available for GABAergic transmission in awake mice, which represents a mechanism for modulation of the inhibitory tone during sustained neuronal activity.

Astrocyte-Derived Exosomes Contribute to Pathologies of Neuromyelitis Optica Spectrum Disorder in Rodent Model.

OBJECTIVE: Neuromyelitis optica spectrum disorder (NMOSD) is an inflammatory demyelinating disease that leads to severe disability. A large proportion of NMOSD patients are seropositive for aquaporin-4 autoantibodies (AQP4-IgG, named as NMO-IgG) targeting AQP4, which is selectively expressed on astrocytes in the central nervous system. This study tests the hypothesis that in response to NMO-IgG, the pathogenic astrocyte-derived exosomes are released and injure the neighboring cells. METHODS: IgG purified from serum of either NMOSD patients or healthy controls was used to generate astrocyte-derived exosomes (AST-ExosNMO vs AST-ExosCON ) in cultured rat astrocytes. The exosomes were respectively delivered to cultured rat oligodendrocytes in vitro, tissue culture of rat optic nerve ex vivo, and rat optic nerve in vivo to evaluate the pathogenic roles of AST-ExosNMO . The microRNA (miRNA) sequencing of AST-Exos and verification were performed to identify the key pathogenic miRNA. The custom-designed adeno-associated virus (AAV) antagonizing the key miRNA was evaluated for its therapeutic effects in vivo. Moreover, the serum levels of the key exosomal miRNA were measured between NMOSD patients and healthy controls. RESULTS: AST-ExosNMO led to notable demyelination in both cultured oligodendrocytes and optic nerve tissue. Exosomal miR-129-2-3p was identified as the key miRNA mediating the demyelinating pathogenesis via downstream target gene SMAD3. AAV antagonizing miR-129-2-3p protected against demyelination in an NMOSD rodent model. The serum exosomal miR-129-2-3p level was significantly elevated in NMOSD patients and correlated with disease severity. INTERPRETATION: Astrocytes targeted by NMO-IgG release pathogenic exosomes that could potentially be used as therapeutic targets or disease monitoring biomarkers in NMOSD. ANN NEUROL 2023;94:163-181.

Correlation of Circadian Rhythms of Heart Rate Variability Indices with Stress, Mood, and Sleep Status in Female Medical Workers with Night Shifts.

Purpose: Heart rate variability (HRV) indices have been used as stress indicators. Rare studies investigated the associations of circadian rhythms of the HRV indices with the stress, mood, and sleep conditions in populations under stress. Methods: In total 257 female participants (203 shift workers and 54 non-shift workers) were included. All the participants completed a structured questionnaire to assess the stress, mood, and sleep conditions and performed 24-hour Holter electrocardiogram monitoring on the day away from shifts. Using epochs of 1-min or 5-min beat-to-beat intervals, the HRV indices (SDNN, RMSSD, LF, HF, LF/HF, and LFnu, SD1, SD2, SD1/SD2) were plotted as a function of time and fitted into cosine periodic curves, respectively. Three mathematical parameters based on the cosine periodic curves were extracted, MESOR (M, overall averages of the cosine curve), amplitude (A, amplitude of the peak of the cosine curve), and acrophase (θ, latency to the peak) to quantify the circadian rhythms of the HRV indices. Multivariable linear regression models were used to reveal the associations of these parameters with the clinical assessments of stress, mood, or sleep conditions, as well as with the 24-h averages of the HRV indices. Results: The parameters M and A of SDNN, RMSSD, LF, and HF, and θ of LF/HF and LFnu significantly differ between shift and non-shift workers. The parameter θ of LF/HF positively correlates with the severity of stress and anxiety. The parameter A of LF/HF and LFnu also positively correlates with daytime sleepiness and sleep fragmentation. In addition, the parameters M and A instead of θ of SDNN, RMSSD, LF, LF/HF, and LFnu significantly correlate with the 24-h averages of HRV indices. Conclusion: The circadian rhythms of the HRV indices over 24 hours can, to some extent, predict the severity of stress, emotion and sleep conditions in female populations under stress.

Optic nerve head: A gatekeeper for vitreous infectious insults?

The axons of retinal ganglion cells (RGCs) pass through the optic nerve head (ONH) and form the optic nerve (ON). The ONH serves as an anatomical interface between the vitreous cavity and subarachnoid space. After inducing acute neuroinflammation by intravitreal injection of lipopolysaccharides (LPS), we observed inflammatory activation in the retina, but detect no signs of inflammation in the posterior ON or infiltration of inflammatory cells in the ONH. Therefore, we hypothesized that the ONH functions as a barrier to vitreous inflammation. Using transmission electron microscopy, we identified significant increase in G-ratio in the posterior ON on day 7 post intravitreal injection (PII) of LPS compared with the phosphate buffered saline (PBS) group. Moreover, using confocal imaging of ex vivo tissue extracted from Aldh1L1-eGFP reporter mice, we observed that the ONH astrocytes altered their spatial orientation by elongating their morphology along the axonal axis of RGCs in LPS- versus PBS-treated eyes; this was quantified by the ratio of longitudinal (DL) and transverse (DT) diameter of astrocytes and the proportion of longitudinally locating astrocytes. Supportive evidences were further provided by transmission electron microscopic imaging in rat ONH. We further conducted RNA sequencing of ONH on day 1 PII and found LPS induced clear upregulation of immune and inflammatory pathways. Furthermore, gene set enrichment analysis revealed that astrocyte and microglia contributed prominently to the transcriptomic alterations in ONH. Here, we report that the vitreous infectious insults induce morphological changes of ONH astrocytes and transcriptomic alterations in the ONH. Glial responses in the ONH may defend against vitreous infectious insults and serve as a barrier to inflammation for the central nervous system.

A visualization pipeline for in vivo two-photon volumetric astrocytic calcium imaging.

Astrocytes, the multi-functional glial cells with the most abundant population in the brain, integrate information across their territories to regulate neuronal synaptic and cerebrovascular activities. Astrocytic calcium (Ca 2+) signaling is the major readout of cellular functional state of astrocytes. The conventional two-photon in vivo imaging usually focuses on a single horizontal focal plane to capture the astrocytic Ca 2+ signals, which leaves >80% spatial information undetected. To fully probe the Ca 2+ activity across the whole astrocytic territory, we developed a pipeline for imaging and visualizing volumetric astrocytic Ca 2+ time-lapse images. With the pipeline, we discovered a new signal distribution pattern from three-dimensional (3D) astrocytic Ca 2+ imaging data of mice under isoflurane anesthetic states. The tools developed in this study enable a better understanding of the spatiotemporal patterns of astrocytic activity in 3D space.

Does Chronic Sleep Fragmentation Lead to Alzheimer's Disease in Young Wild-Type Mice?

Chronic sleep insufficiency is becoming a common issue in the young population nowadays, mostly due to life habits and work stress. Studies in animal models of neurological diseases reported that it would accelerate neurodegeneration progression and exacerbate interstitial metabolic waste accumulation in the brain. In this paper, we study whether chronic sleep insufficiency leads to neurodegenerative diseases in young wild-type animals without a genetic pre-disposition. To this aim, we modeled chronic sleep fragmentation (SF) in young wild-type mice. We detected pathological hyperphosphorylated-tau (Ser396/Tau5) and gliosis in the SF hippocampus. 18F-labeled fluorodeoxyglucose positron emission tomography scan (18F-FDG-PET) further revealed a significant increase in brain glucose metabolism, especially in the hypothalamus, hippocampus and amygdala. Hippocampal RNAseq indicated that immunological and inflammatory pathways were significantly altered in 1.5-month SF mice. More interestingly, differential expression gene lists from stress mouse models showed differential expression patterns between 1.5-month SF and control mice, while Alzheimer's disease, normal aging, and APOEε4 mutation mouse models did not exhibit any significant pattern. In summary, 1.5-month sleep fragmentation could generate AD-like pathological changes including tauopathy and gliosis, mainly linked to stress, as the incremented glucose metabolism observed with PET imaging suggested. Further investigation will show whether SF could eventually lead to chronic neurodegeneration if the stress condition is prolonged in time.

A Chronic Sleep Fragmentation Model using Vibrating Orbital Rotor to Induce Cognitive Deficit and Anxiety-Like Behavior in Young Wild-Type Mice.

Sleep disturbance is generally common in populations as a chronic disease or a complained event. Chronic sleep disturbance is proposed to be closely linked to the pathogenesis of diseases, especially neurodegenerative diseases. We recently found that 2 months of sleep fragmentation initiated Alzheimer's disease (AD)-like behavioral and pathological changes in young wild-type mice. Herein, we present a standardized protocol to achieve chronic sleep fragmentation (CSF). Briefly, CSF was induced by an orbital rotor vibrating at 110 rpm and operating with a repetitive cycle of 10 s-on, 110 s-off, during light-ON phase (8:00 AM-8:00 PM) continuously for up to 2 months. Impairments of spatial learning and memory, anxiety-like but not depression-like behavior in mice as consequences of CSF modeling, were evaluated with Morris water maze (MWM), Novel object recognition (NOR), Open field test (OFT) and Forced swimming test (FST). In comparison with other sleep manipulations, this protocol minimizes the handling labors and maximizes the modeling efficiency. It produces stable phenotypes in young wild-type mice and can be potentially generated for a variety of research purposes.

Chronic sleep fragmentation shares similar pathogenesis with neurodegenerative diseases: Endosome-autophagosome-lysosome pathway dysfunction and microglia-mediated neuroinflammation.

AIMS: Insufficient sleep has been found to result in varying degrees of cognitive impairment and emotional changes. Sleep was reported probably responsible for cleaning metabolic wastes in brain by increasing extracellular bulk flow. Herein, we propose that chronic sleep insufficiency in young adult wild-type mice is also linked with dysfunction of intracellular protein degradation pathways and microglia-mediated neuroinflammation, which are potentially important mechanisms in the initiation of neurodegeneration. METHODS: We applied the chronic sleep fragmentation (CSF) model to induce chronic sleep insufficiency in wild-type mice. After 2 months of CSF, cognitive function, amyloid-ß accumulation, dysfunction of endosome-autophagosome-lysosome pathway, and microglia activation were evaluated. RESULTS: Following CSF, impairment of spatial learning and memory, and aggravated anxiety-like behavior in mice were identified by behavioral experiments. Increased intracellular amyloid-ß accumulation was observed in cortex and hippocampus. Mechanistically, CSF could significantly enhance the expression of Rab5 (early endosome marker), Rab7 (late endosome marker), as well as LC3B (autophagosome marker), and autophagy-positive regulatory factors in brain detected by immunofluorescent staining and Western blot. In addition, activation of microglia was evident by enhanced CD68, CD16/32, and CD206 levels after CSF treatment. CONCLUSIONS: Chronic sleep fragmentation could initiate pathogenetic processes similar to the early stage of neurodegeneration, including dysfunction of endosome-autophagosome-lysosome pathway and microglia-mediated neuroinflammation. Our findings further strengthen the link between chronic sleep insufficiency and the initiation of neurodegeneration even if lack of genetic predisposition.

Focal Solute Trapping and Global Glymphatic Pathway Impairment in a Murine Model of Multiple Microinfarcts.

Microinfarcts occur commonly in the aging brain as a consequence of diffuse embolic events and are associated with the development of vascular dementia and Alzheimer's disease. However, the manner in which disperse microscopic lesions reduce global cognitive function and increase the risk for Alzheimer's disease is unclear. The glymphatic system, which is a brain-wide perivascular network that supports the recirculation of CSF through the brain parenchyma, facilitates the clearance of interstitial solutes including amyloid ß and tau. We investigated whether glymphatic pathway function is impaired in a murine model of multiple microinfarcts induced by intraarterial injection of cholesterol crystals. The analysis showed that multiple microinfarcts markedly impaired global influx of CSF along the glymphatic pathway. Although suppression of global glymphatic function was transient, resolving within 2 weeks of injury, CSF tracers also accumulated within tissue associated with microinfarcts. The effect of diffuse microinfarcts on global glymphatic pathway function was exacerbated in the mice aged 12 months compared with the 2- to 3-month-old mice. These findings indicate that glymphatic function is focally disrupted around microinfarcts and that the aging brain is more vulnerable to this disruption than the young brain. These observations suggest that microlesions may trap proteins and other interstitial solutes within the brain parenchyma, increasing the risk of amyloid plaque formation.SIGNIFICANCE STATEMENT Microinfarcts, small (<1 mm) ischemic lesions, are strongly associated with age-related dementia. However, how these microscopic lesions affect global cognitive function and predispose to Alzheimer's disease is unclear. The glymphatic system is a brain-wide network of channels surrounding brain blood vessels that allows CSF to exchange with interstitial fluid, clearing away cellular wastes such as amyloid ß. We observed that, in mice, microinfarcts impaired global glymphatic function and solutes from the CSF became trapped in tissue associated with microinfarcts. These data suggest that small, disperse ischemic lesions can impair glymphatic function across the brain and trapping of solutes in these lesions may promote protein aggregation and neuroinflammation and eventually lead to neurodegeneration, especially in the aging brain.