Delirium is a common and severe neuropsychiatric syndrome with lasting cognitive consequences, yet its pathophysiology remains poorly understood. We hypothesize that impaired glymphatic flow represents a central mechanism by which delirium evolves. This hypothesis builds on recent evidence showing that major delirium risk factors, such as ageing, dementia, cardiovascular disease and renal failure, are all associated with reduced glymphatic clearance. Similarly, common delirium triggers, including infection, surgery and sleep deprivation, have been shown to impair glymphatic function. In addition, standard intensive care interventions linked to delirium, such as sedation, opioid administration and noradrenaline, are known to suppress glymphatic clearance. Collectively, these effects could lead to the accumulation of neurotoxic metabolites and pro-inflammatory cytokines, disrupting neural network activity and precipitating delirium. Recognizing glymphatic impairment as a causal contributor to delirium could open new research and therapeutic avenues aimed at preserving brain fluid clearance in critically ill patients.

Abstract This study presents a novel in vivo neuroimaging approach using dynamic single photon emission computed tomography (SPECT/CT) to investigate cerebrospinal fluid (CSF) dynamics in pigs, a translationally relevant model due to their human-like brain structure. The distribution, brain penetration of [99mTc]-DTPA and subsequent clearance were followed by brain SPECT for three hours after injection into the cisterna magna of anesthetized pigs and rats. To investigate the effects of anesthesia and across-species effects, we examine CSF dynamics under two types of anesthesia, propofol and ketamine/dexmedetomidine (K/D), and compare the outcome in pigs to that of rats, in which we also compared isoflurane. Propofol and K/D produced largely similar tracer distribution patterns across both pigs and rats: In both species, K/D was associated with higher tracer penetration into the dorsal striatum compared to propofol while neither species showed a tracer accumulation difference in the thalamus. K/D also increased intracranial radiotracer retention and reduced urinary tracer clearance in rats, but not in pigs. In rats, propofol and isoflurane showed similar tracer distribution, reflecting their shared GABAergic mechanism of action. The differences observed between pigs and rats may reflect species-specific physiology, differences in anesthesia dosing, or methodological factors. The work demonstrates the feasibility of using SPECT/CT to study CSF transport in the large gyrencephalic pig brain to advance understanding of human brain fluid dynamics.

The circulation of cerebrospinal fluid (CSF) through perivascular spaces (PVSs) has been proposed to play a role in clearing waste from the brain. While several studies have quantified such flow in mice, no in vivo studies with high spatial and temporal resolution have been performed on other species. Here, we imaged pial PVSs of rat brains and the CSF flows they carry, using techniques previously employed with mice. Using vessel pulsatility measurement and automated particle tracking, we quantified and analyzed in vivo CSF velocity profiles, artery wall motion, and PVS resistances in rats, comparing our findings to mice. Both species exhibit net CSF flows in the same direction as blood flow, and CSF pulsations are substantially synchronized with the heartbeat. We found that rats have larger, lower-resistance PVSs, resulting in much larger CSF volume flow rates in rats than in mice. We also found that the choice of anesthetic can significantly affect vasomotion and corresponding CSF pulsations in rats, with ketamine-dexmedetomidine producing a more stable response than ketamine-xylazine. Our results can be used in models of flow through rat PVSs, and as experiments are performed on additional species, can aid in projecting results across scales from animal models to human CSF flows.

This study investigated whether magnetic resonance (MR) diffusivity parameters derived from diffusion-weighted imaging (DWI) can serve as biomarkers of glymphatic function in awake and anesthetized mice. Spectral apparent diffusion coefficient (ADC) analysis, obtained using an inverse Laplace transform, revealed that both Isoflurane (ISO) and Ketamine/Xylazine (K/X) anesthesia reduced the magnitude and range of diffusivities associated with interstitial fluid space (≤1 µm²/ms) compared with the awake state. Perfusion-related diffusivities (20–80 µm²/ms) increased under ISO but decreased under K/X. Monoexponential ADC and biexponential intravoxel incoherent motion (IVIM) modeling showed that ISO dose-dependently elevated, while K/X reduced, both slow and fast diffusivities across the brain. Dynamic contrast-enhanced MRI (DCE-MRI) indicated intermediate glymphatic influx in awake mice relative to both anesthetic conditions, without regional correlation to DWI-derived parameters. Perfusion micro–computed tomography (µCT) further demonstrated that ADC and IVIM metrics correlated regionally with mean transit time, suggesting a confounding by cerebral blood flow (CBF). Two-photon microscopy confirmed anesthesia-induced changes in cortical microvessel diameters consistent with perfusion alterations. Collectively, these findings indicate that MR diffusivity measures are strongly influenced by state-dependent physiological changes, and in particular perfusion. This represents an important limitation that warrants caution when using DWI to compare extracellular space, interstitial fluid flow, or glymphatic activity across different physiological or anesthetic states. Therefore, although technically challenging, we suggest that DWI studies aimed at assessing glymphatic or interstitial dynamics should be performed in awake conditions to minimize variability and anesthesia-related perfusion confounds across studies.

Sleep is essential for maintaining brain tissue homeostasis, which is facilitated by enhanced cerebrospinal fluid (CSF) solute transport. Infraslow (<0.1 Hz) vasomotion, CSF flow, and electrophysiological potential all increase during sleep, but their contributions as potential drivers of CSF flow in human brain remain unknown. To investigate this, we measured these signals in healthy volunteers across sleep-wake states using functional MRI blood oxygen level-dependent (BOLD), electroencephalography, and functional near-infrared spectroscopy. We then studied the directed coupling patterns between the three signals, using phase transfer entropy. In the awake state, electrophysiological potential and water concentration changes both predicted hemodynamic BOLD changes across whole brain, reflecting classical functional hyperemia. During sleep, these interactions changed such that the net directionality was lost and the interactions became more bidirectional. Our results show that in addition to neural changes during sleep, nonneural processes such as vasomotor-driven hydrodynamic waves start to gain more impact on human brain activity.

The cochlear aqueduct, located within the temporal bone, forms a narrow connection between the cerebrospinal fluid (CSF) in the subarachnoid space and the perilymph of the scala tympani in the inner ear. The anatomical linkage provides a potential interface for pressure regulation, molecular exchange, and therapeutic access to the cochlea. Recent findings further indicate that the cochlear aqueduct is functionally coupled to the brain's glymphatic system, permitting CSF flow to the inner ear and raising the possibility of an inner ear glymphatic system. Morphological studies demonstrate substantial interspecies variability in cochlear aqueduct size and patency among species, and identified a diaphragm-like terminal membrane at its lateral end. In mice, immunocytochemical analyses indicate a lymphatic-like phenotype reminiscent of the subarachnoid lymphatic-like membrane (SLYM). The presence of macrophages with phagocytic capacity and the fact that the membrane is activated during bacterial meningitis further supports a role in local immune surveillance. The translational relevance of the cochlear aqueduct has gained attention with advances in inner-ear gene therapy. Tracers injected in cisterna magna in mice reach the inner ear rapidly. In rodents and non-human primates, intracisternal injection of viral vectors via achieves efficient bilateral cochlear transduction, including restoration of hearing in VGLUT3-deficient mice, a model of nonsyndromic deafness. These findings position the cochlear as a minimally invasive route for inner-ear therapy, potentially avoiding direct cochlear surgery. However, interspecies differences and age-related changes in cochlear aqueduct patency must be carefully evaluated before clinical translation.

An age-related decline in vasodilation mediated by neurovascular nitric oxide (NO) and vasoconstriction driven by the Piezo1 receptor precede the aggregation of soluble brain proteins such as amyloid-β (Aβ), which then causes further disruption of brain solute homeostasis, ultimately leading to neurodegeneration in Alzheimer’s disease. Preclinical studies show that restoring these vascular functions increases neurofluidic efflux and improves cognitive outcomes. Here, we tested effects of sublingual NO and/or Piezo1 receptor-targeted mechanotransductive whole-body vibrations (WBV p ) in healthy adults (n = 29) on brain fluid dynamics and CNS-to-blood protein efflux using multimodal neuroimaging and blood biomarker analysis. The combined vasomechanic interventions (NO+WBV p ) produced a synergistic enhancement of brain fluid transport and markedly increased the efflux of soluble brain-derived proteins (Aβ 40&42 , glial fibrillary acidic protein) into the bloodstream. The effects increase with age and the magnitude of NO-induced hypotension. Importantly, the combined intervention was well-tolerated, with no severe adverse physiological responses. Results demonstrate that a simple, non-invasive vasomechanic intervention can transiently promote brain-to-blood protein clearance in humans, highlighting a potentially safe and accessible therapeutic avenue for neurodegenerative conditions characterized by impaired brain solute removal and protein aggregation.

Current evidence suggests that the rejuvenating effects of parabiosis on brain function arise from the exchange of blood factors that enhance synaptic plasticity, promote neurogenesis, and reduce neuroinflammation in aged animals. However, aging is also associated with diminished tissue oxygenation. Here, we report that erythrocytes (red blood cells, RBCs) from aged mice exhibit reduced responsiveness to low oxygen tension (PO2) and release O2 slower than those from young mice. In vivo, sensory stimulation evoked a smaller and delayed capillary RBC flow in aged mice. Although activity-evoked PO2 dips were diminished in aged mice; experimentally reducing PO2 to comparable levels did not restore capillary flow in aged mice, consistent with diminished RBC O2 responsiveness observed ex vivo. Notably, RBCs from aged mice in heterochronic parabiosis pairs (young-aged) displayed faster responses to low PO2 compared to those from aged mice in isochronic pairs (aged-aged). Together, these findings across multiple levels of analysis demonstrate that aging impairs RBC responsiveness to O2 and suggest that improved RBC-mediated O2 delivery contributes to the rejuvenating effects of parabiosis.

We read the article by Pusic Sesar and colleagues [...].

Despite the universal need for sleep across animal species, the biological mechanisms underlying the restorative aspects of sleep remain poorly understood. While sleep architecture is traditionally evaluated using EEG, multiple studies have shown a mismatch between EEG-defined parameters and subjective sleep quality. In particular, slow-wave activity – a hallmark of non-REM (NREM) sleep – does not consistently align with perceptions of sleep depth or subsequent well-being. This discrepancy suggests that core physiological processes beyond neuronal activity contribute to the restorative value of sleep. Recent discoveries have identified the glymphatic system as a brain-wide clearance pathway that facilitates the removal of metabolic waste during sleep. In rodents, glymphatic activity is driven by a complex interplay between norepinephrine oscillations, vascular dynamics, and cerebrospinal fluid (CSF) flow – particularly during NREM sleep. Human imaging studies have revealed parallel signatures, including large-scale CSF pulsations and inverse coupling between blood and CSF volumes during sleep. Disruption of these infraslow dynamics has been observed in conditions such as insomnia, chronic fatigue, and sleep misperception, suggesting a potential link between impaired glymphatic function and non-restorative sleep. This review synthesizes the current evidence for glymphatic clearance as a contributor to sleep’s restorative function, discusses emerging biomarkers, such as cyclic alternating patterns (CAP) and pupil-based proxies of noradrenergic tone, and highlights the need for improved methods to evaluate glymphatic function in humans. We propose that brain clearance may represent a key physiological determinant of restorative sleep and suggest future directions to test this hypothesis across health and disease.

Treatment of neurological diseases that involve oligodendrocytes or astrocytes would benefit from the selective delivery of viral vectors to their common parent, glial progenitor cells (GPCs). Here, we select adeno-associated virus (AAV) capsids with tropism for human GPCs and demonstrate efficient and widespread delivery of the resultant AAVs throughout the mouse brain through the glymphatic system. In vivo screening of a library of capsid-modified, recombination-reported AAVs in chimeric mice engrafted with PDGFRA-driven Cre recombinase-expressing human GPCs identified a set of AAV5-based vectors that preferentially infect human GPCs and/or their astrocyte and oligodendrocyte progeny in vivo, with minimal systemic infection. To maximize the intracerebral distribution of these vectors while minimizing their dosing and extracerebral spread, we paired intracisternal delivery with systemic hypertonicity to increase glymphatic influx. This method bypasses the blood-brain barrier, delivering AAV directly into the brain parenchyma. Glymphatic delivery of our capsid-modified AAV5s enables efficient transgene delivery to human glia throughout the entire adult mouse brain, with minimal off-target transduction.

During wakefulness, neuromodulators operate largely independently to support behavior and cognition. By contrast, sleep reorganizes their activity into a coordinated brain rhythm. During sleep, the major neuromodulators-norepinephrine, acetylcholine, serotonin, and dopamine-exhibit synchronized fluctuations with a periodicity of ~50 seconds. These oscillations appear as recurrent bursts of fast (10 to 30 hertz) electroencephalography activity and are phase-coupled to cerebrospinal fluid flow. Neuromodulators are vasoactive agents and drive slow vasomotion, which provide the mechanical force that supports glymphatic clearance of metabolic waste. Disruption of neuromodulator signaling, as seen in psychiatric disorders, cardiovascular disease, aging, or with commonly prescribed drugs, impairs clearance of neurotoxic proteins, including amyloid-β and tau. Failure of this evolutionarily conserved brain rhythm may therefore represent a previously unrecognized mechanistic pathway linking diverse disorders with sleep disturbances to increased dementia risk.

The dystrophin associated complex (DAC) is an integral membrane scaffold that regulates cellular polarization and structural organization in both brain and peripheral tissues. In the brain, the DAC anchors the water channel aquaporin 4 (AQP4) to astrocytic vascular endfeet, thereby supporting glymphatic waste clearance during the inactive phase. Recent studies have raised the question of whether DAC gene expression is under circadian control. By mining four independent circadian transcriptome and translatome databases, covering multiple brain regions, peripheral tissues, ages, and species, we show that both Aqp4 and the DAC components exhibit circadian rhythms in gene expression across most of the brain and body in mice, chickens and baboons. In addition, Aqp1 shows circadian rhythmicity in peripheral tissues. The phase of these rhythms varies by tissue type yet collectively supports the hypothesis that clock-regulated DAC activity represents a conserved mechanism for coordinating ion, water, and structural homeostasis throughout the body.

Abstract Retinopathy is a common symptom in mitochondrial diseases, and a leading cause of blindness in working-age individuals, often arising as a consequence of diabetes. Here, we demonstrate that postnatal loss of the replicative helicase of mitochondrial DNA in the astrocytes and Müller glia induces neovascular retinopathy. In these retinas, the macroglia show pathological reactivation, leading to hallmark features of neovascularization with blood-retina-barrier leakage, secondary microgliosis, and complement cascade activation. Similar reactivation of astrocytes in the cerebral cortex does not compromise vascular integrity, indicating tissue-specific roles of mitochondrial metabolism in macroglia for vascular homeostasis. Three secreted angiogenic factors—Fgf2, Pgf, and Lcn2—known to contribute to diabetic retinopathy, were induced. Spike recordings of the most sensitive retinal ganglion cells revealed normal rod function and intact retinal coding. These findings highlight the critical role of glial mitochondrial metabolism in neovascular retinopathy, with important implications for therapy development for mitochondrial and common forms of vision loss.

The mammalian brain stores glucose, the main circulating energy substrate, as glycogen. In rodents, the cerebellum contains relatively high glycogen levels, yet its cellular and subcellular distribution remains poorly defined. Using monoclonal antibodies against glycogen, we examined its distribution in the mouse cerebellar cortex. Glycogen was predominantly localized to Bergmann glia (BG) processes in the molecular layer and was also detected in Purkinje cells (PCs), the principal cerebellar neurons. To assess the functional significance of cerebellar glycogen, we analyzed behavior in mice lacking glycogen synthase 1 (Gys1) in BG or PCs using a floxed Gys1 line. Gys1 deficiency in either PCs or GFAP-positive cells reduced anxiety-like behavior, whereas combined deletion caused PC degeneration and ataxia. These findings reveal a critical role for glycogen metabolism in both astrocytes and neurons in cerebellar function.

Stroke remains a leading cause of morbidity and mortality worldwide, with few effective interventions to promote recovery. Targeting circadian timing and glymphatic function may represent viable therapeutic strategies. Here, we show that the small-molecule clock modulator, KL001; high-dose melatonin; acute light pulses; and active-phase time-restricted feeding were each sufficient to enhance glymphatic function in mice. Moreover, initiating treatment with either KL001 or active-phase time-restricted feeding 3 days after preclinical models of stroke improved motor outcomes, reduced lesion volume, increased glymphatic flow, and lowered poststroke brain cytokine burden. These findings suggest that reinforcing normal daily rhythmicity after stroke can markedly enhance neurological recovery, even when interventions are initiated several days after stroke onset.

The sleep-wake cycle subdivides brain activity into distinct states of neural circuit activity, fluid transport, and interstitial-volume fraction. We used in vivo two-photon imaging of mice to ask whether cortical astrocytes exhibit state-dependent volume changes. Our analysis showed that the astrocyte volume expands during wakefulness and shrinks during non-rapid eye movement (NREM) sleep and even more during rapid eye movement (REM) sleep. Norepinephrine (NE) exhibited state-dependent fluctuations that were mirrored by the corresponding changes in astrocytic volume. Pharmacologically blockage of α1-adrenergic, but not α2- or β-adrenergic, receptors, resulted in astrocyte shrinkage. Both chemogenetic and optogenetic stimulation of tyrosine hydroxylase (TH)-positive neurons in the locus coeruleus (LC) significantly increased cortical astrocytic cell volume, which was abolished by the α1-receptor antagonist prazosin. We propose that astrocytic expansion during wakefulness, driven by NE leads to a corresponding shrinkage of the interstitial-volume fraction, increasing neuroglia interactions and suppressing glymphatic flow.

Autonomic regulation of heart and respiratory rates is essential for understanding brain-body interactions in health and disease. Preclinical cardiovascular recordings are often performed under anesthesia or via telemetry, both of which introduce physiological confounds such as stress or impaired recovery due to the need for acute or chronic implantation of sensors. Here, we present a minimally invasive protocol for simultaneous acquisition of high-quality electrocardiography and respiratory signals in awake mice. Using an in-house-modified physiological monitor in awake, head-fixed mice that were briefly habituated to experimental conditions, we ultimately enable stable, long-term physiological recordings alongside in vivo microscopy. This protocol provides a robust, low-stress method for acquiring physiological signals, enabling the simultaneous study of cardiovascular-cerebral dynamics in awake head-fixed mice, thereby enhancing the translational relevance of preclinical measurements. Key features • Minimally invasive electrocardiogram and respiration rate acquisition in awake, head-fixed mice suitable for long-term physiological recordings. • Custom-built setup integrates physiological monitoring with in vivo imaging without surgical implantation or telemetry. • Rapid habituation protocol ensures low-stress conditions and high-quality signal acquisition in conscious mice. • Enables correlation of cardiovascular dynamics with brain activity and cerebrospinal fluid flow in translational neuroscience studies.

PurposeFluid flow and transport of therapeutic agents and pathogenic bacteria into the cochlea has been challenging to study due to its small size and location within bone. We here take advantage of recent non-invasive Computed Tomography (CT) imaging to infer transport parameters in a 1-dimensional advection-diffusion model of the cochlear aqueduct using a Bayesian approach.MethodsSix male C56BL/6 mice injected with the small molecule tracer iohexol in cisterna magna were scanned every 5 min for 30 min in CT under anesthesia. Using the CT data to set boundary conditions, we solve the advection-diffusion equation for given advection and spatially varying diffusion parameters. The CT data is modeled as normally distributed around the model-predicted concentration. We specify priors for the model unknown parameters and infer their posterior distributions using Bayes' formula and the CUQIpy library. The statistical approach is validated using synthetic data.ResultsThe evolution of the concentration of tracer in the cochlear aqueduct is well predicted by the advection-diffusion model using inferred parameters. Though diffusion of the small CT tracer varies along the aqueduct, it is usually near the free diffusion and therefore not likely to be influenced much by membranes or flow. Advection is inferred near zero in most cases. We show how to use these results to calculate transport through the cochlear aqueduct for other animals and molecules.ConclusionFree diffusion dominates transport of small molecules in the cochlear aqueduct, which can therefore be effectively approximated using simple 1-dimensional (advection-)diffusion formulas.