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.

Structural characterization of SLYM-a 4th meningeal membrane.

Traditionally, the meninges are described as 3 distinct layers, dura, arachnoid and pia. Yet, the classification of the connective meningeal membranes surrounding the brain is based on postmortem macroscopic examination. Ultrastructural and single cell transcriptome analyses have documented that the 3 meningeal layers can be subdivided into several distinct layers based on cellular characteristics. We here re-examined the existence of a 4th meningeal membrane, Subarachnoid Lymphatic-like Membrane or SLYM in Prox1-eGFP reporter mice. Imaging of freshly resected whole brains showed that SLYM covers the entire brain and brain stem and forms a roof shielding the subarachnoid cerebrospinal fluid (CSF)-filled cisterns and the pia-adjacent vasculature. Thus, SLYM is strategically positioned to facilitate periarterial influx of freshly produced CSF and thereby support unidirectional glymphatic CSF transport. Histological analysis showed that, in spinal cord and parts of dorsal cortex, SLYM fused with the arachnoid barrier layer, while in the basal brain stem typically formed a 1-3 cell layered membrane subdividing the subarachnoid space into two compartments. However, great care should be taken when interpreting the organization of the delicate leptomeningeal membranes in tissue sections. We show that hyperosmotic fixatives dehydrate the tissue with the risk of shrinkage and dislocation of these fragile membranes in postmortem preparations.

Structural characterization of SLYM - a 4th meningeal membrane.

Traditionally, the meninges are described as 3 distinct layers, dura, arachnoid and pia. Yet, the classification of the connective meningeal membranes surrounding the brain is based on postmortem macroscopic examination. Ultrastructural and single cell transcriptome analyses have documented that the 3 meningeal layers can be subdivided into several distinct layers based on cellular characteristics. We here re-examined the existence of a 4th meningeal membrane, Subarachnoid Lymphatic-like Membrane or SLYM in Prox1-eGFP reporter mice. Imaging of freshly resected whole brains showed that SLYM covers the entire brain and brain stem and forms a roof shielding the subarachnoid cerebrospinal fluid (CSF)-filled cisterns and the pia-adjacent vasculature. Thus, SLYM is strategically positioned to facilitate periarterial influx of freshly produced CSF and thereby support unidirectional glymphatic CSF transport. Histological analysis showed that, in spinal cord and parts of dorsal cortex, SLYM fused with the arachnoid barrier layer, while in the basal brain stem typically formed a 1-3 cell layered membrane subdividing the subarachnoid space into two compartments. However, great care should be taken when interpreting the organization of the delicate leptomeningeal membranes in tissue sections. We show that hyperosmotic fixatives dehydrate the tissue with the risk of shrinkage and dislocation of these fragile membranes in postmortem preparations.

Structural characterization of SLYM - a 4 th meningeal membrane.

Traditionally, the meninges are described as 3 distinct layers, dura, arachnoid and pia. Yet, the classification of the connective meningeal membranes surrounding the brain is based on postmortem macroscopic examination. Ultrastructural and single cell transcriptome analyses have documented that the 3 meningeal layers can be subdivided into several distinct layers based on cellular characteristics. We here re-examined the existence of a 4 th meningeal membrane, S ubarachnoid Ly mphatic-like M embrane or SLYM in Prox1-eGFP reporter mice. Imaging of freshly resected whole brains showed that SLYM covers the entire brain and brain stem and forms a roof shielding the subarachnoid cerebrospinal fluid (CSF)-filled cisterns and the pia-adjacent vasculature. Thus, SLYM is strategically positioned to facilitate periarterial influx of freshly produced CSF and thereby support unidirectional glymphatic CSF transport. Histological analysis showed that, in spinal cord and parts of dorsal cortex, SLYM fused with the arachnoid barrier layer, while in the basal brain stem typically formed a 1-3 cell layered membrane subdividing the subarachnoid space into two compartments. However, great care should be taken when interpreting the organization of the delicate leptomeningeal membranes in tissue sections. We show that hyperosmotic fixatives dehydrate the tissue with the risk of shrinkage and dislocation of these fragile membranes in postmortem preparations.

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.

A mesothelium divides the subarachnoid space into functional compartments.

The central nervous system is lined by meninges, classically known as dura, arachnoid, and pia mater. We show the existence of a fourth meningeal layer that compartmentalizes the subarachnoid space in the mouse and human brain, designated the subarachnoid lymphatic-like membrane (SLYM). SLYM is morpho- and immunophenotypically similar to the mesothelial membrane lining of peripheral organs and body cavities, and it encases blood vessels and harbors immune cells. Functionally, the close apposition of SLYM with the endothelial lining of the meningeal venous sinus permits direct exchange of small solutes between cerebrospinal fluid and venous blood, thus representing the mouse equivalent of the arachnoid granulations. The functional characterization of SLYM provides fundamental insights into brain immune barriers and fluid transport.

A real-time in vivo clearance assay for quantification of glymphatic efflux.

Glymphatic fluid transport eliminates metabolic waste from the brain including amyloid-ß, yet the methodology for studying efflux remains rudimentary. Here, we develop a method to evaluate glymphatic real-time clearance. Efflux of Direct Blue 53 (DB53, also T-1824 or Evans Blue) injected into the striatum is quantified by imaging the DB53 signal in the vascular compartment, where it is retained due to its high affinity to albumin. The DB53 signal is detectable as early as 15 min after injection and the efflux kinetics are sharply reduced in mice lacking the water channel aquaporin 4 (AQP4). Pharmacokinetic modeling reveal that DB53 efflux is consistent with the existence of two efflux paths, one with fast kinetics (T1/2 = 50 min) and another with slow kinetics (T1/2 = 240 min), in wild-type mice. This in vivo methodology will aid in defining the physiological variables that drive efflux, as well as the impact of brain states or disorders on clearance kinetics.

Dense core vesicle markers in CSF and cortical tissues of patients with Alzheimer's disease.

BACKGROUND: New fluid biomarkers for Alzheimer's disease (AD) that reveal synaptic and neural network dysfunctions are needed for clinical practice and therapeutic trial design. Dense core vesicle (DCV) cargos are promising cerebrospinal fluid (CSF) indicators of synaptic failure in AD patients. However, their value as biomarkers has not yet been determined. METHODS: Immunoassays were performed to analyze the secretory proteins prohormone convertases PC1/3 and PC2, carboxypeptidase E (CPE), secretogranins SgIII and SgII, and Cystatin C in the cerebral cortex (n = 45, provided by Bellvitge University Hospital) and CSF samples (n = 66, provided by The Sant Pau Initiative on Neurodegeneration cohort) from AD patients (n = 56) and age-matched controls (n = 55). RESULTS: In AD tissues, most DCV proteins were aberrantly accumulated in dystrophic neurites and activated astrocytes, whereas PC1/3, PC2 and CPE were also specifically accumulated in hippocampal granulovacuolar degeneration bodies. AD individuals displayed an overall decline of secretory proteins in the CSF. Interestingly, in AD patients, the CSF levels of prohormone convertases strongly correlated inversely with those of neurodegeneration markers and directly with cognitive impairment status. CONCLUSIONS: These results demonstrate marked alterations of neuronal-specific prohormone convertases in CSF and cortical tissues of AD patients. The neuronal DCV cargos are biomarker candidates for synaptic dysfunction and neurodegeneration in AD.

Circadian control of brain glymphatic and lymphatic fluid flow.

The glymphatic system is a network of perivascular spaces that promotes movement of cerebrospinal fluid (CSF) into the brain and clearance of metabolic waste. This fluid transport system is supported by the water channel aquaporin-4 (AQP4) localized to vascular endfeet of astrocytes. The glymphatic system is more effective during sleep, but whether sleep timing promotes glymphatic function remains unknown. We here show glymphatic influx and clearance exhibit endogenous, circadian rhythms peaking during the mid-rest phase of mice. Drainage of CSF from the cisterna magna to the lymph nodes exhibits daily variation opposite to glymphatic influx, suggesting distribution of CSF throughout the animal depends on time-of-day. The perivascular polarization of AQP4 is highest during the rest phase and loss of AQP4 eliminates the day-night difference in both glymphatic influx and drainage to the lymph nodes. We conclude that CSF distribution is under circadian control and that AQP4 supports this rhythm.

In Vivo Imaging of Cerebrospinal Fluid Transport through the Intact Mouse Skull using Fluorescence Macroscopy.

Cerebrospinal fluid (CSF) flow in rodents has largely been studied using ex vivo quantification of tracers. Techniques such as two-photon microscopy and magnetic resonance imaging (MRI) have enabled in vivo quantification of CSF flow but they are limited by reduced imaging volumes and low spatial resolution, respectively. Recent work has found that CSF enters the brain parenchyma through a network of perivascular spaces surrounding the pial and penetrating arteries of the rodent cortex. This perivascular entry of CSF is a primary driver of the glymphatic system, a pathway implicated in the clearance of toxic metabolic solutes (e.g., amyloid-ß). Here, we illustrate a new macroscopic imaging technique that allows real-time, mesoscopic imaging of fluorescent CSF tracers through the intact skull of live mice. This minimally-invasive method facilitates a multitude of experimental designs and enables single or repeated testing of CSF dynamics. Macroscopes have high spatial and temporal resolution and their large gantry and working distance allow for imaging while performing tasks on behavioral devices. This imaging approach has been validated using two-photon imaging and fluorescence measurements obtained from this technique strongly correlate with ex vivo fluorescence and quantification of radio-labeled tracers. In this protocol, we describe how transcranial macroscopic imaging can be used to evaluate glymphatic transport in live mice, offering an accessible alternative to more costly imaging modalities.

Aquaporin-4-dependent glymphatic solute transport in the rodent brain.

The glymphatic system is a brain-wide clearance pathway; its impairment contributes to the accumulation of amyloid-ß. Influx of cerebrospinal fluid (CSF) depends upon the expression and perivascular localization of the astroglial water channel aquaporin-4 (AQP4). Prompted by a recent failure to find an effect of Aqp4 knock-out (KO) on CSF and interstitial fluid (ISF) tracer transport, five groups re-examined the importance of AQP4 in glymphatic transport. We concur that CSF influx is higher in wild-type mice than in four different Aqp4 KO lines and in one line that lacks perivascular AQP4 (Snta1 KO). Meta-analysis of all studies demonstrated a significant decrease in tracer transport in KO mice and rats compared to controls. Meta-regression indicated that anesthesia, age, and tracer delivery explain the opposing results. We also report that intrastriatal injections suppress glymphatic function. This validates the role of AQP4 and shows that glymphatic studies must avoid the use of invasive procedures.

Amyloid-ß Impairs Vesicular Secretion in Neuronal and Astrocyte Peptidergic Transmission.

Regulated secretion of neuropeptides and neurotrophic factors critically modulates function and plasticity of synapses and circuitries. It is believed that rising amyloid-ß (Aß) concentrations, synaptic dysfunction and network disorganization underlie early phases of Alzheimer's disease (AD). Here, we analyze the impact of soluble Aß1-42 assemblies on peptidergic secretion in cortical neurons and astrocytes. We show that neurons and astrocytes differentially produce and release carboxypeptidase E (CPE) and secretogranin III (SgIII), two dense-core vesicle (DCV) markers belonging to the regulated secretory pathway. Importantly, Aß1-42, but not scrambled Aß1-42, dramatically impairs basal and Ca2+-regulated secretions of endogenously produced CPE and SgIII in cultured neurons and astrocytes. Additionally, KCl-evoked secretion of the DCV cargo brain-derived neurotrophic factor (BDNF) is lowered by Aß1-42 administration, whereas glutamate release from synaptic vesicle (SVs) remains unchanged. In agreement with cell culture results, Aß1-42 effects on CPE and SgIII secretion are faithfully recapitulated in acute adult brain slices. These results demonstrate that neuronal and astrocyte secretion of DCV cargos is impaired by Aß in vitro and in situ. Furthermore, Aß-induced dysregulated peptidergic transmission could have an important role in the pathogenesis of AD and DCV cargos are possible candidates as cerebrospinal fluid (CSF) biomarkers.

Behaviour and cognitive changes correlated with hippocampal neuroinflammaging and neuronal markers in female SAMP8, a model of accelerated senescence.

Senescence accelerated mice P8 (SAMP8) is a phenotypic model of age, characterized by deficits in memory and altered behaviour. Here, we determined the effect of age in SAMP8, and compared with the resistant strain, SAMR1, in behaviour and learning parameters linking these disturbances with oxidative stress environment. We found impairment in emotional behaviour with regard to fear and anxiety in young SAMP8 vs. age-mated SAMR1. Differences were attenuated with age. In contrast, learning capabilities are worse in SAMP8, both in young and aged animals, with regard to SAMR1. These waves in behaviour and cognition were correlated with an excess of oxidative stress (OS) in SAMP8 at younger ages that diminished with age. In this manner, we found changes in the hippocampal expression of ALDH2, IL-6, HMOX1, COX2, CXCL10, iNOS, and MCP-1 with an altered amyloidogenic pathway by increasing the Amyloid beta precursor protein (APP) and BACE1, and reduced ADAM10 expression; in addition, astrogliosis and neuronal markers decreased. Moreover, Superoxide dismutase 1 (SOD1) and Nuclear factor-kappa beta (NF-kß) expression and protein levels were higher in younger SAMP8 than in SAMR1. In conclusion, the accelerated senescence process present in SAMP8 can be linked with an initial deregulation in redox homeostasis, named neuroinflammaging, by inducing molecular changes that lead to neuroinflammation and the neurodegenerative process. These changes are reflected in the emotional and cognitive behaviour of SAMP8 that differs from that of SAMR1 and that highlighted the importance of earlier oxidative processes in the onset of neurodegeneration.

Cyclic AMP signaling restricts activation and promotes maturation and antioxidant defenses in astrocytes.

BACKGROUND: cAMP signaling produces dramatic changes in astrocyte morphology and physiology. However, its involvement in phenotype acquisition and the transcriptionally mediated mechanisms of action are largely unknown. RESULTS: Here we analyzed the global transcriptome of cultured astroglial cells incubated with activators of cAMP pathways. A bulk of astroglial transcripts, 6221 annotated genes, were differentially regulated by cAMP signaling. cAMP analogs strongly upregulated genes involved in typical functions of mature astrocytes, such as homeostatic control, metabolic and structural support to neurons, antioxidant defense and communication, whereas they downregulated a considerable number of proliferating and immaturity-related transcripts. Moreover, numerous genes typically activated in reactive cells, such as scar components and immunological mediators, were repressed by cAMP. GSEA analysis contrasting gene expression profiles with transcriptome signatures of acutely isolated astrocytes and in situ evaluation of protein levels in these cells showed that cAMP signaling conferred mature and in vivo-like transcriptional features to cultured astrocytes. CONCLUSIONS: These results indicate that cAMP signaling is a key pathway promoting astrocyte maturation and restricting their developmental and activation features. Therefore, a positive modulation of cAMP signaling may promote the normal state of differentiated astrocytes and favor the protection and function of neuronal networks.

Secretory sorting receptors carboxypeptidase E and secretogranin III in amyloid ß-associated neural degeneration in Alzheimer's disease.

The secretory sorting receptors carboxypeptidase E (CPE) and secretogranin III (SgIII) critically activate peptidic messengers and targeting them at the regulated secretory pathway. In Alzheimer's disease (AD), the wide range of changes includes impaired function of key secretory peptidic cargos such as brain-derived neurotrophic factor (BDNF) and neuropeptides. Here, we analyzed CPE and SgIII in the cerebral cortex of AD patients and transgenic mice. In the normal human cortex, a preferential location in dendrites and perikarya was observed for CPE, whereas SgIII was mainly associated with axons and terminal-like buttons. Interestingly, SgIII and CPE were consistently detected in astroglial cell bodies and thin processes. In AD cortices, a strong wide accumulation of both sorting receptors was detected in dystrophic neurites surrounding amyloid plaques. Occasionally, increased levels of SgIII were also observed in plaque associate-reactive astrocytes. Of note, the main alterations detected for CPE and SgIII in AD patients were faithfully recapitulated by APPswe/PS1dE9 mice. These results implicate for the first time the sorting receptors for regulated secretion in amyloid ß-associated neural degeneration. Because CPE and SgIII are essential in the process and targeting of neuropeptides and neurotrophins, their participation in the pathological progression of AD may be suggested.