Selective deletion of interleukin-1 alpha in microglia regulates neuronal responses and neurorepair processes after experimental ischemic stroke.

Inflammation is a key contributor to stroke pathogenesis and drives exacerbated brain damage leading to poor outcome. Interleukin-1 (IL-1) is an important regulator of post-stroke inflammation, and blocking its actions is beneficial in pre-clinical stroke models and safe in the clinical setting. IL-1α and IL-1ß are the two major IL-1 type 1 receptor (IL-1R1) agonists from the IL-1 family. The distinct roles of both isoforms, and particularly that of IL-1α, remain largely unknown. Here we show that IL-1α and IL-1ß have different spatio-temporal expression profiles in the brain after experimental stroke, with early microglial IL-1α expression (4 h) and delayed IL-1ß expression in infiltrated neutrophils and a small microglial subset (24-72 h). We examined the specific contribution of microglial-derived IL-1α in experimental permanent and transient ischemic stroke through cell-specific tamoxifen-inducible Cre-loxP-mediated recombination. Microglial IL-1α deletion did not influence acute brain damage, cerebral blood flow, IL-1ß expression, neutrophil infiltration, microglial nor endothelial activation after ischemic stroke. However, microglial IL-1α knock out (KO) mice showed reduced peri-infarct vessel density and reactive astrogliosis at 14 days post-stroke, alongside a worse functional recovery. RNA sequencing analysis and subsequent pathway analysis on ipsilateral/contralateral cortex 4 h after stroke revealed a downregulation of the neuronal CREB signaling pathway in microglial IL-1α KO compared to WT mice. Our study identifies for the first time a critical role for microglial IL-1α on neuronal activity, neurorepair and functional recovery after stroke, highlighting the importance of targeting specific IL-1 mechanisms in brain injury to develop more effective therapies.

Improving stroke outcomes in hyperglycemic mice by modulating tPA/NMDAR signaling to reduce inflammation and hemorrhages.

ABSTRACT: The pharmacological intervention for ischemic stroke hinges on intravenous administration of the recombinant tissue-type plasminogen activator (rtPA, Alteplase/Actilyse) either as a standalone treatment or in conjunction with thrombectomy. However, despite its clinical significance, broader use of rtPA is constrained because of the risk of hemorrhagic transformations (HTs). Furthermore, the presence of diabetes or chronic hyperglycemia is associated with an elevated risk of HT subsequent to thrombolysis. This detrimental impact of tPA on the neurovascular unit in patients with hyperglycemia has been ascribed to its capacity to induce endothelial N-methyl-D-aspartate receptor (NMDAR) signaling, contributing to compromised blood-brain barrier integrity and neuroinflammatory processes. In a mouse model of thromboembolic stroke with chronic hyperglycemia, we assessed the effectiveness of rtPA and N-acetylcysteine (NAC) as thrombolytic agents. We also tested the effect of blocking tPA/NMDAR signaling using a monoclonal antibody, Glunomab. Magnetic resonance imaging, speckle contrast imaging, flow cytometry, and behavioral tasks were used to evaluate stroke outcomes. In hyperglycemic animals, treatment with rtPA resulted in lower recanalization rates and increased HTs. Conversely, NAC treatment reduced lesion sizes while mitigating HTs. After a single administration, either in standalone or combined with rtPA-induced thrombolysis, Glunomab reduced brain lesion volumes, HTs, and neuroinflammation after stroke, translating into improved neurological outcomes. Additionally, we demonstrated the therapeutic efficacy of Glunomab in combination with NAC or as a standalone strategy in chronic hyperglycemic animals. Counteracting tPA-dependent endothelial NMDAR signaling limits ischemic damages induced by both endogenous and exogenous tPA, including HTs and inflammatory processes after ischemic stroke in hyperglycemic animals.

Consequences of General Anesthesia in Infancy on Behavior and Brain Structure.

BACKGROUND: One in 7 children will need general anesthesia (GA) before the age of 3. Brain toxicity of anesthetics is controversial. Our objective was to clarify whether exposure of GA to the developing brain could lead to lasting behavioral and structural brain changes. METHODS: A first study was performed in mice. The behaviors (fear conditioning, Y-maze, and actimetry) and brain anatomy (high-resolution magnetic resonance imaging) of 6- to 8-week-old Swiss mice exposed or not exposed to GA from 4 to 10 days old were evaluated. A second study was a complementary analysis from the preexisting APprentissages EXécutifs et cerveau chez les enfants d'âge scolaire (APEX) cohort to assess the replicability of our data in humans. The behaviors (behavior rating inventory of executive function, emotional control, and working memory score, Backward Digit Span, and Raven 36) and brain anatomy (high-resolution magnetic resonance imaging) were compared in 102 children 9 to 10 years of age exposed or not exposed to a single GA (surgery) during infancy. RESULTS: The animal study revealed chronic exacerbated fear behavior in the adult mice (95% confidence interval [CI], 4-80; P = .03) exposed to postnatal GA; this was associated with an 11% (95% CI, 7.5-14.5) reduction of the periaqueductal gray matter (P = .046). The study in humans suggested lower emotional control (95% CI, 0.33-9.10; P = .06) and a 6.1% (95% CI, 4.3-7.8) reduction in the posterior part of the right inferior frontal gyrus (P = .019) in the children who had been exposed to a single GA procedure. CONCLUSIONS: The preclinical and clinical findings of these independent studies suggest lasting effects of early life exposure to anesthetics on later emotional control behaviors and brain structures.

Selective brain entry of lipid nanoparticles in haemorrhagic stroke is linked to biphasic blood-brain barrier disruption.

Haemorrhagic stroke represents a significant public health burden, yet our knowledge and ability to treat this type of stroke are lacking. Previously we showed that we can target ischaemic-stroke lesions by selective translocation of lipid nanoparticles through the site of blood-brain barrier (BBB) disruption. The data we presented in this study provide compelling evidence that haemorrhagic stroke in mice induces BBB injury that mimics key features of the human pathology and, more importantly, provides a gate for entry of lipid nanoparticles-based therapeutics selectively to the bleeding site. Methods: Haemorrhagic stroke was induced in mice by intra-striatal collagenase injection. lipid nanoparticles were injected intravenously at 3 h, 24 h & 48 h post-haemorrhagic stroke and accumulation in the brain studied using in-vivo optical imaging and histology. BBB integrity, brain water content and iron accumulation were characterised using dynamic contrast-enhanced MRI, quantitative T1 mapping, and gradient echo MRI. Results: Using in-vivo SPECT/CT imaging and optical imaging revealed biphasic lipid nanoparticles entry into the bleeding site, with an early phase of increased uptake at 3-24 h post-haemorrhagic stroke, followed by a second phase at 48-72 h. Lipid nanoparticles entry into the brain post-haemorrhage showed an identical entry pattern to the trans-BBB leakage rate (Ktrans [min-1]) of Gd-DOTA, a biomarker for BBB disruption, measured using dynamic contrast-enhanced MRI. Discussion: Our findings suggest that selective accumulation of liposomes into the lesion site is linked to a biphasic pattern of BBB hyper-permeability. This approach provides a unique opportunity to selectively and efficiently deliver therapeutic molecules across the BBB, an approach that has not been utilised for haemorrhagic stroke therapy and is not achievable using free small drug molecules.

PKCδ-positive GABAergic neurons in the central amygdala exhibit tissue-type plasminogen activator: role in the control of anxiety.

Tissue plasminogen activator (tPA) is a serine protease expressed in several brain regions and reported to be involved in the control of emotional and cognitive functions. Nevertheless, little is known about the structure-function relationships of these tPA-dependent behaviors. Here, by using a new model of constitutive tPA-deficient mice (tPAnull), we first show that tPA controls locomotor activity, spatial cognition and anxiety. To investigate the brain structures involved in these tPA-dependent behavioral phenotypes, we next generated tPAflox mice allowing conditional tPA deletion (cKO) following stereotaxic injections of adeno-associated virus driving Cre-recombinase expression (AAV-Cre-GFP). We demonstrate that tPA removal in the dentate gyrus of the hippocampus induces hyperactivity and partial spatial memory deficits. Moreover, the deletion of tPA in the central nucleus of the amygdala, but not in the basolateral nucleus, induces hyperactivity and reduced anxiety-like level. Importantly, we prove that these behaviors depend on the tPA present in the adult brain and not on neurodevelopmental disorders. Also, interestingly, our data show that tPA from Protein kinase-C delta-positive (PKCδ) GABAergic interneurons of the lateral/ capsular part of adult mouse central amygdala controls emotional functions through neuronal activation of the medial central amygdala. Together, our study brings new data about the critical central role of tPA in behavioral modulations in adult mice.

Itaconate and fumarate derivatives inhibit priming and activation of the canonical NLRP3 inflammasome in macrophages.

The NLRP3 inflammasome is a multiprotein complex that regulates caspase-1 activation and subsequent interleukin (IL)-1ß and IL-18 release from innate immune cells in response to infection or injury. Derivatives of the metabolites itaconate and fumarate, dimethyl itaconate (DMI), 4-octyl itaconate (4OI) and dimethyl fumarate (DMF) limit both expression and release of IL-1ß following NLRP3 inflammasome activation. However, the direct effects of these metabolite derivatives on NLRP3 inflammasome responses require further investigation. Using murine bone marrow-derived macrophages, mixed glia and organotypic hippocampal slice cultures (OHSCs), we demonstrate that DMI, 4OI and DMF pretreatments inhibit pro-inflammatory cytokine production in response to lipopolysaccharide (LPS), as well as inhibit subsequent NLRP3 inflammasome activation induced by nigericin. DMI, 4OI, DMF and monomethyl fumarate (MMF), another fumarate derivative, also directly inhibited biochemical markers of NLRP3 activation in LPS-primed macrophages, mixed glia, OHSCs and human macrophages in response to nigericin and imiquimod, including ASC speck formation, caspase-1 activation, gasdermin D cleavage and IL-1ß release. DMF, an approved treatment of multiple sclerosis, as well as DMI, 4OI and MMF, inhibited NLRP3 activation in macrophages in response to lysophosphatidylcholine, which is used to induce demyelination, suggesting a possible mechanism for DMF in multiple sclerosis through NLRP3 inhibition. The derivatives also reduced pro-IL-1α cleavage in response to the calcium ionophore ionomycin. Together, these findings reveal the immunometabolic regulation of both the priming and activation steps of NLRP3 activation in macrophages. Furthermore, we highlight itaconate and fumarate derivatives as potential therapeutic options in NLRP3- and IL-1α-driven diseases, including in the brain.

LRRC8A is dispensable for a variety of microglial functions and response to acute stroke.

Microglia, resident brain immune cells, are critical in orchestrating responses to central nervous system (CNS) injury. Many microglial functions, such as phagocytosis, motility and chemotaxis, are suggested to rely on chloride channels, including the volume-regulated anion channel (VRAC), but studies to date have relied on the use of pharmacological tools with limited specificity. VRAC has also been proposed as a drug target for acute CNS injury, and its role in microglial function is of considerable interest for developing CNS therapeutics. This study aimed to definitively confirm the contribution of VRAC in microglia function by using conditional LRRC8A-knockout mice, which lacked the essential VRAC subunit LRRC8A in microglia. We demonstrated that while VRAC contributed to cell volume regulation, it had no effect on phagocytic activity, cell migration or P2YR12-dependent chemotaxis. Moreover, loss of microglial VRAC did not affect microglial morphology or the extent of ischemic damage following stroke. We conclude that VRAC does not critically regulate microglial responses to brain injury and could be targetable in other CNS cell types (e.g., astrocytes) without impeding microglial function. Our results also demonstrate a role for VRAC in cell volume regulation but show that VRAC is not involved in several major cellular functions that it was previously thought to regulate, and point to other, alternative mechanisms of chloride transport in innate immunity.

Thrombolysis by PLAT/tPA increases serum free IGF1 leading to a decrease of deleterious autophagy following brain ischemia.

Cerebral ischemia is a pathology involving a cascade of cellular mechanisms, leading to the deregulation of proteostasis, including macroautophagy/autophagy, and finally to neuronal death. If it is now accepted that cerebral ischemia induces autophagy, the effect of thrombolysis/energy recovery on proteostasis remains unknown. Here, we investigated the effect of thrombolysis by PLAT/tPA (plasminogen activator, tissue) on autophagy and neuronal death. In two in vitro models of hypoxia reperfusion and an in vivo model of thromboembolic stroke with thrombolysis by PLAT/tPA, we found that ischemia enhances neuronal deleterious autophagy. Interestingly, PLAT/tPA decreases autophagy to mediate neuroprotection by modulating the PI3K-AKT-MTOR pathways both in vitro and in vivo. We identified IGF1R (insulin-like growth factor I receptor; a tyrosine kinase receptor) as the effective receptor and showed in vitro, in vivo and in human stroke patients and that PLAT/tPA is able to degrade IGFBP3 (insulin-like growth factor binding protein 3) to increase IGF1 (insulin-like growth factor 1) bioavailability and thus IGF1R activation.Abbreviations: AKT/protein kinase B: thymoma viral proto-oncogene 1; EGFR: epidermal growth factor receptor; Hx: hypoxia; IGF1: insulin-like growth factor 1; IGF1R: insulin-like growth factor I receptor; IGFBP3: insulin-like growth factor binding protein 3; Ka: Kainate; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAPK/ERK: mitogen-activated protein kinase; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; OGD: oxygen and glucose deprivation; OGDreox: oxygen and glucose deprivation + reoxygentation; PepA: pepstatin A1; PI3K: phosphoinositide 3-kinase; PLAT/tPA: plasminogen activator, tissue; PPP: picropodophyllin; SCH77: SCH772984; ULK1: unc-51 like kinase 1; Wort: wortmannin.

Robust thrombolytic and anti-inflammatory action of a constitutively active ADAMTS13 variant in murine stroke models.

Advances in our understanding of ADAMTS13 structure, and the conformation changes required for full activity, have rejuvenated the possibility of its use as a thrombolytic therapy. We have tested a novel Ala1144Val ADAMTS13 variant (constitutively active [ca] ADAMTS13) that exhibits constitutive activity, characterized using in vitro assays of ADAMTS13 activity, and greatly enhanced thrombolytic activity in 2 murine models of ischemic stroke, the distal FeCl3 middle cerebral artery occlusion (MCAo) model and transient middle cerebral artery occlusion (tMCAO) with systemic inflammation and ischemia/reperfusion injury. The primary measure of efficacy in both models was restoration of regional cerebral blood flow (rCBF) to the MCA territory, which was determined using laser speckle contrast imaging. The caADAMTS13 variant exhibited a constitutively active conformation and a fivefold enhanced activity against fluorescence resonance energy transfer substrate von Willebrand factor 73 (FRETS-VWF73) compared with wild-type (wt) ADAMTS13. Moreover, caADAMTS13 inhibited VWF-mediated platelet capture at subphysiological concentrations and enhanced t-PA/plasmin lysis of fibrin(ogen), neither of which were observed with wtADAMTS13. Significant restoration of rCBF and reduced lesion volume was observed in animals treated with caADAMTS13. When administered 1 hour after FeCl3 MCAo, the caADAMTS13 variant significantly reduced residual VWF and fibrin deposits in the MCA, platelet aggregate formation, and neutrophil recruitment. When administered 4 hours after reperfusion in the tMCAo model, the caADAMTS13 variant induced a significant dissolution of platelet aggregates and a reduction in the resulting tissue hypoperfusion. The caADAMTS13 variant represents a potentially viable therapeutic option for the treatment of acute ischemic stroke, among other thrombotic indications, due to its enhanced in vitro and in vivo activities that result from its constitutively active conformation.

Two-Chains Tissue Plasminogen Activator Unifies Met and NMDA Receptor Signalling to Control Neuronal Survival.

Tissue-type plasminogen activator (tPA) plays roles in the development and the plasticity of the nervous system. Here, we demonstrate in neurons, that by opposition to the single chain form (sc-tPA), the two-chains form of tPA (tc-tPA) activates the MET receptor, leading to the recruitment of N-Methyl-d-Aspartate receptors (NMDARs) and to the endocytosis and proteasome-dependent degradation of NMDARs containing the GluN2B subunit. Accordingly, tc-tPA down-regulated GluN2B-NMDAR-driven signalling, a process prevented by blockers of HGFR/MET and mimicked by its agonists, leading to a modulation of neuronal death. Thus, our present study unmasks a new mechanism of action of tPA, with its two-chains form mediating a crosstalk between MET and the GluN2B subunit of NMDARs to control neuronal survival.

Hallmarks of NLRP3 inflammasome activation are observed in organotypic hippocampal slice culture.

Microglial inflammation driven by the NACHT, LRR and PYD domain-containing protein 3 (NLRP3) inflammasome contributes to brain disease and is a therapeutic target. Most mechanistic studies on NLRP3 activation use two-dimensional pure microglial cell culture systems. Here we studied the activation of the NLRP3 inflammasome in organotypic hippocampal slices, which allowed us to investigate microglial NLRP3 activation in a three-dimensional, complex tissue architecture. Toll-like receptor 2 and 4 activation primed microglial inflammasome responses in hippocampal slices by increasing NLRP3 and interleukin-1ß expression. Nigericin-induced NLRP3 inflammasome activation was dynamically visualized in microglia through ASC speck formation. Downstream caspase-1 activation, gasdermin D cleavage, pyroptotic cell death and interleukin-1ß release were also detected, and these findings were consistent when using different NLRP3 stimuli such as ATP and imiquimod. NLRP3 inflammasome pathway inhibitors were effective in organotypic hippocampal slices. Hence, we have highlighted organotypic hippocampal slice culture as a valuable ex vivo tool to allow the future study of NLRP3 inflammasomes in a representative tissue section, aiding the discovery of further mechanistic insights and drug development.

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.

Ligature-induced periodontitis induces systemic inflammation but does not alter acute outcome after stroke in mice.

BACKGROUND: Stroke is a major cause of disability and mortality. Poorer outcome after stroke is associated with concomitant inflammatory and infectious disease. Periodontitis is a chronic inflammatory disease of the dental supporting structures and is a prominent risk factor for many systemic disorders, including cardiovascular disease and stroke. While epidemiological studies suggest that periodontitis increases the likelihood of stroke, its impact on stroke severity is poorly understood. Here, we sought to determine the contribution of periodontitis to acute stroke pathology. METHODS: We characterized a murine ligature model of periodontitis for inflammatory responses that could potentially impact stroke outcome. We applied this model and then subjected mice to either transient or permanent middle cerebral artery occlusion. We also enhanced the periodontitis model with repeated intravenous administration of a periodontal-specific lipopolysaccharide to better mimic the clinical condition. RESULTS: Ligature-induced periodontitis caused bone loss, bacterial growth, and increased local inflammatory cell trafficking. Systemically, periodontitis increased circulating levels of pro-inflammatory cytokines, and primed bone marrow monocytes to produce elevated tumour necrosis factor-alpha (TNFα). Despite these changes, periodontitis alone or in tandem with repeated lipopolysaccharide challenge did not alter infarct volume, blood-brain barrier breakdown, or systemic inflammation after experimental stroke. CONCLUSIONS: Our data show that despite elevated systemic inflammation in periodontitis, oral inflammatory disease does not impact acute stroke pathology in terms of severity, determined primarily by infarct volume. This indicates that, at least in this experimental paradigm, periodontitis alone does not alter acute outcome after cerebral ischemia.

Pentraxin 3 regulates neutrophil infiltration to the brain during neuroinflammation.

Introduction: The acute phase protein pentraxin 3 (PTX3) is known for its anti-inflammatory effects through downregulating neutrophil transmigration during peripheral inflammation. Furthermore, we have previously demonstrated a neuroprotective and neuroreparative effect of PTX3 after cerebral ischaemia. Here we investigated, to our knowledge for the first time, the role of PTX3 in neutrophil transmigration and neurotoxicity following lipopolysaccharide (LPS)-induced cerebral inflammation and cerebral ischaemia. Methods: Neutrophil transmigration through interleukin-1ß (IL-1ß) activated brain endothelium and neurotoxicity of neutrophils isolated from wild-type (WT) or PTX3 knock-out (KO) mice was assessed in vitro. Primary cortical neuronal death after treatment with transmigrated neutrophils was quantified by lactate dehydrogenase (LDH) assay. Cerebral inflammation or ischemia was induced in WT and PTX3 KO mice via intrastriatal LPS injection or by transient middle cerebral artery occlusion (MCAo) respectively. Subsequent neutrophil infiltration in the brain was assessed by immunohistochemistry and the expression of pro-inflammatory cytokines interleukin-6 (IL-6) and IL-1ß by enzyme-linked immunosorbent assay (ELISA). Results: Neutrophils isolated from WT mice after intrastriatal LPS injection transmigrated significantly more through IL-1ß activated brain endothelium compared to neutrophils from PTX3 KO mice. Transmigrated WT and PTX3 KO neutrophils were significantly more neurotoxic than corresponding non-transmigrated neutrophils; however, no significant differences in neurotoxicity between genotypes were observed. PTX3 reduced the number of transmigrated neutrophils to the brain after intrastriatal LPS injection. Furthermore, PTX3 KO mice showed significantly increased levels of neutrophils in the brain after LPS administration or in the ischaemic hemisphere after MCAo, compared to WT mice. Conclusion: Our study shows that PTX3 regulates neutrophil transmigration in the CNS during neuroinflammation, demonstrating the potential of PTX3 as an effective therapeutic target in neuroinflammatory conditions.

Extent of Ischemic Brain Injury After Thrombotic Stroke Is Independent of the NLRP3 (NACHT, LRR and PYD Domains-Containing Protein 3) Inflammasome.

Background and Purpose- A major process contributing to cell death in the ischemic brain is inflammation. Inflammasomes are multimolecular protein complexes that drive inflammation through activation of proinflammatory cytokines, such as IL (interleukin)-1ß. Preclinical evidence suggests that IL-1ß contributes to a worsening of ischemic brain injury. Methods- Using a mouse middle cerebral artery thrombosis model, we examined the inflammatory response after stroke and the contribution of the NLRP3 (NACHT, LRR and PYD domains-containing protein 3) inflammasome to ischemic injury. Results- There was a marked inflammatory response after stroke characterized by increased expression of proinflammatory cytokines and NLRP3 and by recruitment of leukocytes to the injured tissue. Targeting NLRP3 with the inhibitor MCC950, or using mice in which NLRP3 was knocked out, had no effect on the extent of injury caused by stroke. Conclusions- These data suggest that the NLRP3 pathway does not contribute to the inflammation exacerbating ischemic brain damage, contradicting several recent reports to the contrary.

Small, Thin Graphene Oxide Is Anti-inflammatory Activating Nuclear Factor Erythroid 2-Related Factor 2 via Metabolic Reprogramming.

Graphene oxide (GO), an oxidized form of graphene, has potential applications in biomedical research. However, how GO interacts with biological systems, including the innate immune system, is poorly understood. Here, we elucidate the effects of GO sheets on macrophages, identifying distinctive effects of GO on the inflammatory phenotype. Small, thin (s)-GO dose-dependently inhibited release of interleukin (IL)-1ß and IL-6 but not tumor necrosis factor α. NLRP3 inflammasome and caspase-1 activation was not affected. The effect of s-GO was pretranslational, as s-GO blocked Toll-like receptor 4-dependent expression of Il1b and Il6 but not Nlrp3 or Tnf mRNA transcripts. s-GO was internalized by immortalized bone-marrow-derived macrophages, suggesting a potential intracellular action. Uptake of polystyrene beads with similar lateral dimensions and surface charge did not phenocopy the effects of s-GO, suggesting that s-GO-mediated inhibition of interleukin expression was not simply due to particle phagocytosis. RNA-Seq analysis established that s-GO had profound effects on the immunometabolism of the cells, leading to activation of the transcription factor nuclear factor erythroid 2-related factor 2, which inhibited expression of cytokines such as IL-1ß and IL-6. Thus, we have identified immunometabolic effects of GO that reveal another dimension to its effects on cells. These findings suggest that s-GO may be used as a valuable tool to generate further insights into inflammatory mechanisms and indicate its potential applications in biomedicine.

Pentraxin 3 promotes long-term cerebral blood flow recovery, angiogenesis, and neuronal survival after stroke.

Restoration of cerebral blood flow (CBF) and upregulation of angiogenesis are crucial for brain repair and functional recovery after cerebral ischaemia. Pentraxin 3 (PTX3) is a key regulator of angiogenesis and is emerging as a promising target for cerebrovascular repair after stroke. Here, we investigated for the first time the role of PTX3 in long-term CBF, angiogenesis, and neuronal viability after ischaemic stroke induced by transient middle cerebral artery occlusion (MCAo). Lack of PTX3 had no effect on early brain damage, but significantly impaired restoration of CBF, 14 and 28 days after MCAo, compared to wild-type (WT) mice. Immunohistochemical analysis revealed that PTX3 KO mice have significantly greater neuronal loss, significantly decreased vessel diameter, vessel proliferation, vascular density, and reactive astrocytes and decreased expression of vascular endothelial growth factor receptor 2 (VEGR2), vascular extracellular matrix (ECM)-proteins (collagen IV, laminin), and integrin-ß, in the ipsilateral (stroke) hemisphere compared to WT mice, 28 days after MCAo. Therefore, PTX3 promotes sustained long-term recovery of CBF, angiogenesis, and neuronal viability after cerebral ischaemia. Collectively, these findings demonstrate the potential and clinical relevance of PTX3 as a promising therapeutic target, providing sustained long-term post-stroke neurovascular repair and reducing the loss of neurons. KEY MESSAGES: Pentraxin 3 (PTX3) is a key regulator of angiogenesis and is emerging as a promising target for cerebrovascular repair after stroke. Restoration of cerebral blood flow (CBF) and angiogenesis are crucial for brain repair and functional recovery after cerebral ischaemia. PTX3 promotes sustained long-term recovery of CBF, angiogenesis, and neuronal viability after cerebral ischaemia.

Activation of cell surface GRP78 decreases endoplasmic reticulum stress and neuronal death.

The unfolded protein response (UPR) is an endoplasmic reticulum (ER) -related stress conserved pathway that aims to protect cells from being overwhelmed. However, when prolonged, UPR activation converts to a death signal, which relies on its PERK-eIF2α branch. Overactivation of the UPR has been implicated in many neurological diseases, including cerebral ischaemia. Here, by using an in vivo thromboembolic model of stroke on transgenic ER stress-reporter mice and neuronal in vitro models of ischaemia, we demonstrate that ischaemic stress leads to the deleterious activation of the PERK branch of the UPR. Moreover, we show that the serine protease tissue-type plasminogen activator (tPA) can bind to cell surface Grp78 (78 kD glucose-regulated protein), leading to a decrease of the PERK pathway activation, thus a decrease of the deleterious factor CHOP, and finally promotes neuroprotection. Altogether, this work highlights a new role and a therapeutic potential of the chaperone protein Grp78 as a membrane receptor of tPA capable to prevent from ER stress overactivation.

A brain in flame; do inflammasomes and pyroptosis influence stroke pathology?

Stroke is one of the leading causes of death and disability worldwide. Inflammation plays a key role across the time course of stroke, from onset to the post-injury reparative phase days to months later. Several regulatory molecules are implicated in inflammation, but the most established inflammatory mediator of acute brain injury is the cytokine interleukin-1. Interleukin-1 is regulated by large, macromolecular complexes called inflammasomes, which play a central role in cytokine release and cell death. In this review we highlight recent advances in inflammasome research and propose key roles for inflammasome components in the progression of stroke damage.

Impact of alcohol consumption on the outcome of ischemic stroke and thrombolysis: role of the hepatic clearance of tissue-type plasminogen activator.

BACKGROUND AND PURPOSE: Tissue-type plasminogen activator (tPA) is the only acute treatment for ischemic stroke. Unfortunately, the benefit of tPA-driven thrombolysis is not systematic, and understanding the reasons for this is mandatory. The balance between beneficial and detrimental effects of tPA might explain the limited overall efficiency of thrombolysis. Here, we investigated whether this balance could be influenced by excessive alcohol intake. METHODS: We used a murine model of thromboembolic stroke, coupled to an array of biochemical assays, near-infrared or magnetic resonance imaging scans, 2-photon microscopy, hydrodynamic transfections, and immunohistological techniques. RESULTS: We found that 6 weeks of alcohol consumption (10% in drinking water) worsens ischemic lesions and cancels the beneficial effects of tPA-induced thrombolysis. We accumulate in vivo and in vitro evidence showing that this aggravation is correlated with a decrease in lipoprotein receptor-related protein 1-mediated hepatic clearance of tPA in alcohol-exposed mice. CONCLUSIONS: An efficient liver-driven clearance of tPA might influence the safety of thrombolysis after stroke.