Oxygen imaging of hypoxic pockets in the mouse cerebral cortex.

Consciousness is lost within seconds upon cessation of cerebral blood flow. The brain cannot store oxygen, and interruption of oxidative phosphorylation is fatal within minutes. Yet only rudimentary knowledge exists regarding cortical partial oxygen tension (Po2) dynamics under physiological conditions. Here we introduce Green enhanced Nano-lantern (GeNL), a genetically encoded bioluminescent oxygen indicator for Po2 imaging. In awake behaving mice, we uncover the existence of spontaneous, spatially defined "hypoxic pockets" and demonstrate their linkage to the abrogation of local capillary flow. Exercise reduced the burden of hypoxic pockets by 52% compared with rest. The study provides insight into cortical oxygen dynamics in awake behaving animals and concurrently establishes a tool to delineate the importance of oxygen tension in physiological processes and neurological diseases.

Overuse of EEG and ECG in children with breath-holding spells and its implication for the management of the spells.

AIM: Breath-holding spells (BHS) are common in children, but evidence-based clinical guidelines are lacking. We investigated a large population-based cohort of BHS patients, to propose a refined description of typical BHS and guidelines for its management. METHODS: In a cross-sectional retrospective study, patients diagnosed with BHS in Southern Sweden 2004-2018 were recruited. Disease characteristics and diagnostic data were collected from patient medical records. RESULTS: In total, 519 patients, mean age at diagnosis 19.8 ± 13.8 months with equal gender distribution, were included. In 48.3%, BHS had already been diagnosed after one spell. During spells, 78.0% of patients were unresponsive. For 71.5%, atonic, tonic, tonic-clonic or myoclonic seizures were reported, and 78.0% of patients had a spell lasting less than 1 min. Electroencephalography was conducted in 30.4% and Electrocardiography in 45.1%. Six children (3.8%) had a pathological electroencephalogram, four of which had concomitant epilepsy and only 0.9% of children had electrocardiogram findings suggesting pathology, none showing long QT syndrome. CONCLUSION: Children with BHS were frequently subjected to unnecessary diagnostic interventions. We characterise a typical presentation of BHS and propose a management-algorithm, which is expected to reduce unnecessary usage of electroencephalography and electrocardiography.

Potentiating glymphatic drainage minimizes post-traumatic cerebral oedema.

Cerebral oedema is associated with morbidity and mortality after traumatic brain injury (TBI)1. Noradrenaline levels are increased after TBI2-4, and the amplitude of the increase in noradrenaline predicts both the extent of injury5 and the likelihood of mortality6. Glymphatic impairment is both a feature of and a contributor to brain injury7,8, but its relationship with the injury-associated surge in noradrenaline is unclear. Here we report that acute post-traumatic oedema results from a suppression of glymphatic and lymphatic fluid flow that occurs in response to excessive systemic release of noradrenaline. This post-TBI adrenergic storm was associated with reduced contractility of cervical lymphatic vessels, consistent with diminished return of glymphatic and lymphatic fluid to the systemic circulation. Accordingly, pan-adrenergic receptor inhibition normalized central venous pressure and partly restored glymphatic and cervical lymphatic flow in a mouse model of TBI, and these actions led to substantially reduced brain oedema and improved functional outcomes. Furthermore, post-traumatic inhibition of adrenergic signalling boosted lymphatic export of cellular debris from the traumatic lesion, substantially reducing secondary inflammation and accumulation of phosphorylated tau. These observations suggest that targeting the noradrenergic control of central glymphatic flow may offer a therapeutic approach for treating acute TBI.

Age- and glaucoma-induced changes to the ocular glymphatic system.

The ocular glymphatic system supports bidirectional fluid transport along the optic nerve, thereby removes metabolic wastes including amyloid-ß. To better understand this biological process, we examined the distributions of intravitreally and intracisternally infused tracers in full-length optic nerves from different age groups of mice. Aging was linked to globally impaired ocular glymphatic fluid transport, similar to what has seen previously in the brain. Aging also reduced the pupillary responsiveness to light stimulation and abolished light-induced facilitation in anterograde ocular glymphatic flow. In contrast to normal aging, in the DBA/2 J model of glaucoma, we found a pathological increase of glymphatic fluid transport to the anterior optic nerve that was associated with dilation of the perivascular spaces. Thus, aging and glaucoma have fundamentally different effects on ocular glymphatic fluid transport. Manipulation of glymphatic fluid transport might therefore present a new target for the treatment of glaucoma.

Glymphatic influx and clearance are accelerated by neurovascular coupling.

Functional hyperemia, also known as neurovascular coupling, is a phenomenon that occurs when neural activity increases local cerebral blood flow. Because all biological activity produces metabolic waste, we here sought to investigate the relationship between functional hyperemia and waste clearance via the glymphatic system. The analysis showed that whisker stimulation increased both glymphatic influx and clearance in the mouse somatosensory cortex with a 1.6-fold increase in periarterial cerebrospinal fluid (CSF) influx velocity in the activated hemisphere. Particle tracking velocimetry revealed a direct coupling between arterial dilation/constriction and periarterial CSF flow velocity. Optogenetic manipulation of vascular smooth muscle cells enhanced glymphatic influx in the absence of neural activation. We propose that impedance pumping allows arterial pulsatility to drive CSF in the same direction as blood flow, and we present a simulation that supports this idea. Thus, functional hyperemia boosts not only the supply of metabolites but also the removal of metabolic waste.

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.

Glymphatic-assisted perivascular brain delivery of intrathecal small gold nanoparticles.

Nanoparticles are ultrafine particulate matter having considerable potential for treatment of central nervous system (CNS) disorders. Despite their tiny size, the blood-brain barrier (BBB) restricts their access to the CNS. Their direct cerebrospinal fluid (CSF) administration bypasses the BBB endothelium, but still fails to give adequate brain uptake. We present a novel approach for efficient CNS delivery of 111In-radiolabelled gold nanoparticles (AuNPs; 10-15 nm) via intra-cisterna magna administration, with tracking by SPECT imaging. To accelerate CSF brain influx, we administered AuNPs intracisternally in conjunction with systemic hypertonic saline, which dramatically increased the parenchymal AuNP uptake, especially in deep brain regions. AuNPs entered the CNS along periarterial spaces as visualized by MRI of gadolinium-labelled AuNPs and were cleared from brain within 24 h and excreted through the kidneys. Thus, the glymphatic-assisted perivascular network augment by systemic hypertonic saline is a pathway for highly efficient brain-wide distribution of small AuNPs.

A SPECT-based method for dynamic imaging of the glymphatic system in rats.

The glymphatic system is a brain-wide waste drainage system that promotes cerebrospinal fluid circulation through the brain to remove waste metabolites. Currently, the most common methods for assessing glymphatic function are ex vivo fluorescence microscopy of brain slices, macroscopic cortical imaging, and MRI. While all these methods have been crucial for expanding our understanding of the glymphatic system, new techniques are required to overcome their specific drawbacks. Here, we evaluate SPECT/CT imaging as a tool to assess glymphatic function in different anesthesia-induced brain states using two radiolabeled tracers, [111In]-DTPA and [99mTc]-NanoScan. Using SPECT, we confirmed the existence of brain state-dependent differences in glymphatic flow and we show brain state-dependent differences of CSF flow kinetics and CSF egress to the lymph nodes. We compare SPECT and MRI for imaging glymphatic flow and find that the two imaging modalities show the same overall pattern of CSF flow, but that SPECT was specific across a greater range of tracer concentrations than MRI. Overall, we find that SPECT imaging is a promising tool for imaging the glymphatic system, and that qualities such as high sensitivity and the variety of available tracers make SPECT imaging a good alternative for glymphatic research.

SPECT/CT imaging reveals CNS-wide modulation of glymphatic cerebrospinal fluid flow by systemic hypertonic saline.

Intrathecal administration enables central nervous system delivery of drugs that do not bypass the blood-brain barrier. Systemic administration of hypertonic saline (HTS) enhances delivery of intrathecal therapeutics into the neuropil, but its effect on solute clearance from the brain remains unknown. Here, we developed a dynamic in vivo single-photon emission computed tomography (SPECT)/computed tomography (CT) imaging platform to study the effects of HTS on whole-body distribution of the radiolabeled tracer 99mTc-diethylenetriaminepentaacetic acid (DTPA) administered through intracisternal, intrastriatal, or intravenous route in anesthetized rats. Co-administration of systemic HTS increased intracranial exposure to intracisternal 99mTc-DTPA by ∼80% during imaging. In contrast, HTS had minimal effects on brain clearance of intrastriatal 99mTc-DTPA. In sum, SPECT/CT imaging presents a valuable approach to study glymphatic drug delivery. Using this methodology, we show that systemic HTS increases intracranial availability of cerebrospinal fluid-administered tracer, but has marginal effects on brain clearance, thus substantiating a simple, yet effective strategy for enhancing intrathecal drug delivery to the brain.

Memory-enhancing properties of sleep depend on the oscillatory amplitude of norepinephrine.

Sleep has a complex micro-architecture, encompassing micro-arousals, sleep spindles and transitions between sleep stages. Fragmented sleep impairs memory consolidation, whereas spindle-rich and delta-rich non-rapid eye movement (NREM) sleep and rapid eye movement (REM) sleep promote it. However, the relationship between micro-arousals and memory-promoting aspects of sleep remains unclear. In this study, we used fiber photometry in mice to examine how release of the arousal mediator norepinephrine (NE) shapes sleep micro-architecture. Here we show that micro-arousals are generated in a periodic pattern during NREM sleep, riding on the peak of locus-coeruleus-generated infraslow oscillations of extracellular NE, whereas descending phases of NE oscillations drive spindles. The amplitude of NE oscillations is crucial for shaping sleep micro-architecture related to memory performance: prolonged descent of NE promotes spindle-enriched intermediate state and REM sleep but also associates with awakenings, whereas shorter NE descents uphold NREM sleep and micro-arousals. Thus, the NE oscillatory amplitude may be a target for improving sleep in sleep disorders.

Mapping of CSF transport using high spatiotemporal resolution dynamic contrast-enhanced MRI in mice: Effect of anesthesia.

PURPOSE: Dynamic contrast-enhanced MRI (DCE-MRI) represents the only available approach for glymphatic cerebrospinal fluid (CSF) flow 3D mapping in the brain of living animals and humans. The purpose of this study was to develop a novel DCE-MRI protocol for mapping of the glymphatic system transport with improved spatiotemporal resolution, and to validate the new protocol by comparing the transport in mice anesthetized with either isoflurane or ketamine/xylazine. METHODS: The contrast agent, gadobutrol, was administered into the CSF of the cisterna magna and its transport visualized continuously on a 9.4T preclinical scanner using 3D fast-imaging with a steady-state free-precession sequence (3D-FISP), which has a spatial resolution of 0.001 mm3 and a temporal resolution of 30 s. The MR signals were measured dynamically for 60 min in multiple volumes of interest covering the entire CSF space and brain parenchyma. RESULTS: The results confirm earlier findings that glymphatic CSF influx is higher under ketamine/xylazine than with isoflurane anesthesia. This was extended to account for new details about the distinct CSF efflux pathways under the two anesthetic regimens. Dynamic contrast MR shows that CSF clearance occurs mainly along the vagus nerve near the jugular vein under isoflurane and via the olfactory bulb under ketamine/xylazine. CONCLUSION: The improved spatial and temporal sampling rates afforded by 3D-FISP shed new light on the pharmacological modulation of CSF efflux paths. The present observations may have the potential to set a new standard for future experimental DCE-MRI studies of the glymphatic system.

A three-dimensional, population-based average of the C57BL/6 mouse brain from DAPI-stained coronal slices.

Fluorescence imaging of immunolabeled brain slices is a key tool in neuroscience that enable mapping of proteins or DNA/RNA at resolutions not possible with non-invasive techniques, including magnetic resonance or nuclear imaging. The signal in specific regions is usually quantified after manually drawing regions of interest, risking operator-bias. Automated segmentation methods avoid this risk but require multi-sample average atlases with similar image contrast as the images to be analyzed. We here present the first population-based average atlas of the C57BL/6 mouse brain constructed from brain sections labeled with the fluorescence nuclear stain DAPI. The data set constitutes a rich three-dimensional representation of the average mouse brain in the DAPI staining modality reconstructed from coronal slices and includes an automatic segmentation/spatial normalization pipeline for novel coronal slices. It constitutes the final population-based average template, individual reconstructed brain volumes, and native coronal slices. The comprehensive data set and accompanying spatial normalization/segmentation software are provided. We encourage the community to utilize it to improve and validate methods for automated brain slice analysis.

An ocular glymphatic clearance system removes ß-amyloid from the rodent eye.

Despite high metabolic activity, the retina and optic nerve head lack traditional lymphatic drainage. We here identified an ocular glymphatic clearance route for fluid and wastes via the proximal optic nerve in rodents. ß-amyloid (Aß) was cleared from the retina and vitreous via a pathway dependent on glial water channel aquaporin-4 (AQP4) and driven by the ocular-cranial pressure difference. After traversing the lamina barrier, intra-axonal Aß was cleared via the perivenous space and subsequently drained to lymphatic vessels. Light-induced pupil constriction enhanced efflux, whereas atropine or raising intracranial pressure blocked efflux. In two distinct murine models of glaucoma, Aß leaked from the eye via defects in the lamina barrier instead of directional axonal efflux. The results suggest that, in rodents, the removal of fluid and metabolites from the intraocular space occurs through a glymphatic pathway that might be impaired in glaucoma.

PDGF-B Is Required for Development of the Glymphatic System.

The glymphatic system is a highly polarized cerebrospinal fluid (CSF) transport system that facilitates the clearance of neurotoxic molecules through a brain-wide network of perivascular pathways. Herein we have mapped the development of the glymphatic system in mice. Perivascular CSF transport first emerges in hippocampus in newborn mice, and a mature glymphatic system is established in the cortex at 2 weeks of age. Formation of astrocytic endfeet and polarized expression of aquaporin 4 (AQP4) consistently coincided with the appearance of perivascular CSF transport. Deficiency of platelet-derived growth factor B (PDGF-B) function in the PDGF retention motif knockout mouse line Pdgfbret/ret suppressed the development of the glymphatic system, whose functions remained suppressed in adulthood compared with wild-type mice. These experiments map the natural development of the glymphatic system in mice and define a critical role of PDGF-B in the development of perivascular CSF transport.

Increased glymphatic influx is correlated with high EEG delta power and low heart rate in mice under anesthesia.

The glymphatic system is responsible for brain-wide delivery of nutrients and clearance of waste via influx of cerebrospinal fluid (CSF) alongside perivascular spaces and through the brain. Glymphatic system activity increases during sleep or ketamine/xylazine (K/X) anesthesia, yet the mechanism(s) facilitating CSF influx are poorly understood. Here, we correlated influx of a CSF tracer into the brain with electroencephalogram (EEG) power, heart rate, blood pressure, and respiratory rate in wild-type mice under six different anesthesia regimens. We found that glymphatic CSF tracer influx was highest under K/X followed by isoflurane (ISO) supplemented with dexmedetomidine and pentobarbital. Mice anesthetized with α-chloralose, Avertin, or ISO exhibited low CSF tracer influx. This is the first study to show that glymphatic influx correlates positively with cortical delta power in EEG recordings and negatively with beta power and heart rate.