Neuronal dynamics direct cerebrospinal fluid perfusion and brain clearance.

The accumulation of metabolic waste is a leading cause of numerous neurological disorders, yet we still have only limited knowledge of how the brain performs self-cleansing. Here we demonstrate that neural networks synchronize individual action potentials to create large-amplitude, rhythmic and self-perpetuating ionic waves in the interstitial fluid of the brain. These waves are a plausible mechanism to explain the correlated potentiation of the glymphatic flow1,2 through the brain parenchyma. Chemogenetic flattening of these high-energy ionic waves largely impeded cerebrospinal fluid infiltration into and clearance of molecules from the brain parenchyma. Notably, synthesized waves generated through transcranial optogenetic stimulation substantially potentiated cerebrospinal fluid-to-interstitial fluid perfusion. Our study demonstrates that neurons serve as master organizers for brain clearance. This fundamental principle introduces a new theoretical framework for the functioning of macroscopic brain waves.

Identification of direct connections between the dura and the brain.

The arachnoid barrier delineates the border between the central nervous system and dura mater. Although the arachnoid barrier creates a partition, communication between the central nervous system and the dura mater is crucial for waste clearance and immune surveillance1,2. How the arachnoid barrier balances separation and communication is poorly understood. Here, using transcriptomic data, we developed transgenic mice to examine specific anatomical structures that function as routes across the arachnoid barrier. Bridging veins create discontinuities where they cross the arachnoid barrier, forming structures that we termed arachnoid cuff exit (ACE) points. The openings that ACE points create allow the exchange of fluids and molecules between the subarachnoid space and the dura, enabling the drainage of cerebrospinal fluid and limited entry of molecules from the dura to the subarachnoid space. In healthy human volunteers, magnetic resonance imaging tracers transit along bridging veins in a similar manner to access the subarachnoid space. Notably, in neuroinflammatory conditions such as experimental autoimmune encephalomyelitis, ACE points also enable cellular trafficking, representing a route for immune cells to directly enter the subarachnoid space from the dura mater. Collectively, our results indicate that ACE points are a critical part of the anatomy of neuroimmune communication in both mice and humans that link the central nervous system with the dura and its immunological diversity and waste clearance systems.

Parenchymal border macrophages regulate tau pathology and tau-mediated neurodegeneration.

Parenchymal border macrophages (PBMs) reside close to the central nervous system parenchyma and regulate CSF flow dynamics. We recently demonstrated that PBMs provide a clearance pathway for amyloid-ß peptide, which accumulates in the brain in Alzheimer's disease (AD). Given the emerging role for PBMs in AD, we explored how tau pathology affects the CSF flow and the PBM populations in the PS19 mouse model of tau pathology. We demonstrated a reduction of CSF flow, and an increase in an MHCII+PBM subpopulation in PS19 mice compared with WT littermates. Consequently, we asked whether PBM dysfunction could exacerbate tau pathology and tau-mediated neurodegeneration. Pharmacological depletion of PBMs in PS19 mice led to an increase in tau pathology and tau-dependent neurodegeneration, which was independent of gliosis or aquaporin-4 depolarization, essential for the CSF-ISF exchange. Together, our results identify PBMs as novel cellular regulators of tau pathology and tau-mediated neurodegeneration.

Parenchymal border macrophages regulate the flow dynamics of the cerebrospinal fluid.

Macrophages are important players in the maintenance of tissue homeostasis1. Perivascular and leptomeningeal macrophages reside near the central nervous system (CNS) parenchyma2, and their role in CNS physiology has not been sufficiently well studied. Given their continuous interaction with the cerebrospinal fluid (CSF) and strategic positioning, we refer to these cells collectively as parenchymal border macrophages (PBMs). Here we demonstrate that PBMs regulate CSF flow dynamics. We identify a subpopulation of PBMs that express high levels of CD163 and LYVE1 (scavenger receptor proteins), closely associated with the brain arterial tree, and show that LYVE1+ PBMs regulate arterial motion that drives CSF flow. Pharmacological or genetic depletion of PBMs led to accumulation of extracellular matrix proteins, obstructing CSF access to perivascular spaces and impairing CNS perfusion and clearance. Ageing-associated alterations in PBMs and impairment of CSF dynamics were restored after intracisternal injection of macrophage colony-stimulating factor. Single-nucleus RNA sequencing data obtained from patients with Alzheimer's disease (AD) and from non-AD individuals point to changes in phagocytosis, endocytosis and interferon-γ signalling on PBMs, pathways that are corroborated in a mouse model of AD. Collectively, our results identify PBMs as new cellular regulators of CSF flow dynamics, which could be targeted pharmacologically to alleviate brain clearance deficits associated with ageing and AD.

Persistent neuroinflammation and behavioural deficits after single mild traumatic brain injury.

Despite an apparently silent imaging, some patients with mild traumatic brain injury (TBI) experience cognitive dysfunctions, which may persist chronically. Brain changes responsible for these dysfunctions are unclear and commonly overlooked. It is thus crucial to increase our understanding of the mechanisms linking the initial event to the functional deficits, and to provide objective evidence of brain tissue alterations underpinning these deficits. We first set up a murine model of closed-head controlled cortical impact, which provoked persistent cognitive and sensorimotor deficits, despite no evidence of brain contusion or bleeding on MRI, thus recapitulating features of mild TBI. Molecular MRI for P-selectin, a key adhesion molecule, detected no sign of cerebrovascular inflammation after mild TBI, as confirmed by immunostainings. By contrast, in vivo PET imaging with the TSPO ligand [18F]DPA-714 demonstrated persisting signs of neuroinflammation in the ipsilateral cortex and hippocampus after mild TBI. Interestingly, immunohistochemical analyses confirmed these spatio-temporal profiles, showing a robust parenchymal astrogliosis and microgliosis, at least up to 3 weeks post-injury in both the cortex and hippocampus. In conclusion, we show that even one single mild TBI induces long-term behavioural deficits, associated with a persistent neuro-inflammatory status that can be detected by PET imaging.

Skull and vertebral bone marrow are myeloid cell reservoirs for the meninges and CNS parenchyma.

The meninges are a membranous structure enveloping the central nervous system (CNS) that host a rich repertoire of immune cells mediating CNS immune surveillance. Here, we report that the mouse meninges contain a pool of monocytes and neutrophils supplied not from the blood but by adjacent skull and vertebral bone marrow. Under pathological conditions, including spinal cord injury and neuroinflammation, CNS-infiltrating myeloid cells can originate from brain borders and display transcriptional signatures distinct from their blood-derived counterparts. Thus, CNS borders are populated by myeloid cells from adjacent bone marrow niches, strategically placed to supply innate immune cells under homeostatic and pathological conditions. These findings call for a reinterpretation of immune-cell infiltration into the CNS during injury and autoimmunity and may inform future therapeutic approaches that harness meningeal immune cells.

Functional characterization of the dural sinuses as a neuroimmune interface.

Despite the established dogma of central nervous system (CNS) immune privilege, neuroimmune interactions play an active role in diverse neurological disorders. However, the precise mechanisms underlying CNS immune surveillance remain elusive; particularly, the anatomical sites where peripheral adaptive immunity can sample CNS-derived antigens and the cellular and molecular mediators orchestrating this surveillance. Here, we demonstrate that CNS-derived antigens in the cerebrospinal fluid (CSF) accumulate around the dural sinuses, are captured by local antigen-presenting cells, and are presented to patrolling T cells. This surveillance is enabled by endothelial and mural cells forming the sinus stromal niche. T cell recognition of CSF-derived antigens at this site promoted tissue resident phenotypes and effector functions within the dural meninges. These findings highlight the critical role of dural sinuses as a neuroimmune interface, where brain antigens are surveyed under steady-state conditions, and shed light on age-related dysfunction and neuroinflammatory attack in animal models of multiple sclerosis.

Single- and two- chain tissue type plasminogen activator treatments differentially influence cerebral recovery after stroke.

Tissue type Plasminogen Activator (tPA), named alteplase (Actilyse®) under its commercial form, is currently the only pharmacological treatment approved during the acute phase of ischemic stroke, used either alone or combined with thrombectomy. Interestingly, the commercial recombinant tPA (rtPA) contains two physiological forms of rtPA: the single chain rtPA (sc-rtPA) and the two-chains rtPA (tc-rtPA), with differential properties demonstrated in vitro. Using a relevant mouse model of thromboembolic stroke, we have investigated the overall effects of these two forms of rtPA when infused early after stroke onset (i.e. 20 min) on recanalization, lesion volumes, alterations of the integrity of the blood brain barrier and functional recovery. Our data reveal that there is no difference in the capacity of sc-rtPA and tc-rtPA to promote fibrinolysis and reperfusion of the tissue. However, compared to sc-rtPA, tc-rtPA is less efficient to reduce lesion volumes and to improve functional recovery, and is associated with an increased opening of the blood brain barrier. These data indicate better understanding of differential effects of these tPA forms might be important to ultimately improve stroke treatment.

Alcohol exposure-induced neurovascular inflammatory priming impacts ischemic stroke and is linked with brain perivascular macrophages.

Alcohol abuse is a major public health problem worldwide, causing a wide range of preventable morbidity and mortality. In this translational study, we show that heavy drinking (HD) (≥6 standard drinks/day) is independently associated with a worse outcome for ischemic stroke patients. To study the underlying mechanisms of this deleterious effect of HD, we performed an extensive analysis of the brain inflammatory responses of mice chronically exposed or not to 10% alcohol before and after ischemic stroke. Inflammatory responses were analyzed at the parenchymal, perivascular, and vascular levels by using transcriptomic, immunohistochemical, in vivo 2-photon microscopy and molecular MRI analyses. Alcohol-exposed mice show, in the absence of any other insult, a neurovascular inflammatory priming (i.e., an abnormal inflammatory status including an increase in brain perivascular macrophages [PVM]) associated with exacerbated inflammatory responses after a secondary insult (ischemic stroke or LPS challenge). Similar to our clinical data, alcohol-exposed mice showed larger ischemic lesions. We show here that PVM are key players on this aggravating effect of alcohol, since their specific depletion blocks the alcohol-induced aggravation of ischemic lesions. This study opens potentially new therapeutic avenues aiming at blocking alcohol-induced exacerbation of the neurovascular inflammatory responses triggered after ischemic stroke.

Immune Responses and Anti-inflammatory Strategies in a Clinically Relevant Model of Thromboembolic Ischemic Stroke with Reperfusion.

The poor clinical relevance of experimental models of stroke contributes to the translational failure between preclinical and clinical studies testing anti-inflammatory molecules for ischemic stroke. Here, we (i) describe the time course of inflammatory responses triggered by a thromboembolic model of ischemic stroke and (ii) we examine the efficacy of two clinically tested anti-inflammatory drugs: Minocycline or anti-CD49d antibodies (tested in stroke patients as Natalizumab) administered early (1 h) or late (48 h) after stroke onset. Radiological (lesion volume) and neurological (grip test) outcomes were evaluated at 24 h and 5 days after stroke. Immune cell responses peaked 48 h after stroke onset. Myeloid cells (microglia/macrophages, dendritic cells, and neutrophils) were already increased 24 h after stroke onset, peaked at 48 h, and remained increased-although to a lesser extent-5 days after stroke onset. CD8+ and CD4+ T-lymphocytes infiltrated the ipsilateral hemisphere later on (only from 48 h). These responses occurred together with a progressive blood-brain barrier leakage at the lesion site, starting 24 h after stroke onset. Lesion volume was maximal 24-48 h after stroke onset. Minocycline reduced both lesion volume and neurological deficit only when administered early after stroke onset. The blockade of leukocyte infiltration by anti-CD49d had no impact on lesion volume or long-term neurological deficit, independently of the timing of treatment. Our data are in accordance with the results of previous clinical reports on the use of Minocycline and Natalizumab on ischemic stroke. We thus propose the use of this clinically relevant model of thromboembolic stroke with recanalization for future testing of anti-inflammatory strategies for stroke.

Circulating tPA contributes to neurovascular coupling by a mechanism involving the endothelial NMDA receptors.

The increase of cerebral blood flow evoked by neuronal activity is essential to ensure enough energy supply to the brain. In the neurovascular unit, endothelial cells are ideally placed to regulate key neurovascular functions of the brain. Nevertheless, some outstanding questions remain about their exact role neurovascular coupling (NVC). Here, we postulated that the tissue-type plasminogen activator (tPA) present in the circulation might contribute to NVC by a mechanism dependent of its interaction with endothelial N-Methyl-D-Aspartate Receptor (NMDAR). To address this question, we used pharmacological and genetic approaches to interfere with vascular tPA-dependent NMDAR signaling, combined with laser speckle flowmetry, intravital microscopy and ultrafast functional ultrasound in vivo imaging. We found that the tPA present in the blood circulation is capable of potentiating the cerebral blood flow increase induced by the activation of the mouse somatosensorial cortex, and that this effect is mediated by a tPA-dependent activation of NMDAR expressed at the luminal part of endothelial cells of arteries. Although blood molecules, such as acetylcholine, bradykinin or ATP are known to regulate vascular tone and induce vessel dilation, our present data provide the first evidence that circulating tPA is capable of influencing neurovascular coupling (NVC).

Longitudinal Molecular Magnetic Resonance Imaging of Endothelial Activation after Severe Traumatic Brain Injury.

Traumatic brain injury (TBI) is a major cause of death and disability. Despite progress in neurosurgery and critical care, patients still lack a form of neuroprotective treatment that can counteract or attenuate injury progression. Inflammation after TBI is a key modulator of injury progression and neurodegeneration, but its spatiotemporal dissemination is only partially known. In vivo approaches to study post-traumatic inflammation longitudinally are pivotal for monitoring injury progression/recovery and the effectiveness of therapeutic approaches. Here, we provide a minimally invasive, highly sensitive in vivo molecular magnetic resonance imaging (MRI) characterization of endothelial activation associated to neuroinflammatory response after severe TBI in mice, using microparticles of iron oxide targeting P-selectin (MPIOs-α-P-selectin). Strong endothelial activation was detected from 24 h in perilesional regions, including the cortex and hippocampus, and peaked in intensity and diffusion at two days, then partially decreased but persisted up to seven days and was back to baseline 15 days after injury. There was a close correspondence between MPIOs-α-P-selectin signal voids and the P-selectin stained area, confirming maximal endothelial activation at two days. Molecular MRI markers of inflammation may thus represent a useful tool to evaluate in vivo endothelial activation in TBI and monitoring the responses to therapeutic agents targeting vascular activation and permeability.

Anti-inflammatory treatments for stroke: from bench to bedside.

So far, intravenous tissue-type plasminogen activator (tPA) and mechanical removal of arterial blood clot (thrombectomy) are the only available treatments for acute ischemic stroke. However, the short therapeutic window and the lack of specialized stroke unit care make the overall availability of both treatments limited. Additional agents to combine with tPA administration or thrombectomy to enhance efficacy and improve outcomes associated with stroke are needed. Stroke-induced inflammatory processes are a response to the tissue damage due to the absence of blood supply but have been proposed also as key contributors to all the stages of the ischemic stroke pathophysiology. Despite promising results in experimental studies, inflammation-modulating treatments have not yet been translated successfully into the clinical setting. This review will (a) describe the timing of the stroke immune pathophysiology; (b) detail the immune responses to stroke sift-through cell type; and

Modification of apparent intracerebral hematoma volume on T2∗-weighted images during normobaric oxygen therapy may contribute to false diagnosis.

It was previously reported that normobaric oxygen therapy (NBO) significantly affected T2∗-weighted imaging in a mouse model of intracerebral hemorrhage (ICH). However, it is unclear whether a similar phenomenon exists in large volume ICH as seen in human pathology. We investigated the effects of NBO on T2∗-weighted images in a pig model of ICH. Our data show that NBO makes disappear a peripheral crown of the hematoma, which in turn decreases the apparent volume of ICH by 18%. We hypothesized that this result could be translated to ICH in human, and subsequently could lead to inaccurate diagnostic.