Myeloid cells in vascular dementia and Alzheimer's disease: Possible therapeutic targets?

Growing evidence supports the suggestion that the peripheral immune system plays a role in different pathologies associated with cognitive impairment, such as vascular dementia (VD) or Alzheimer's disease (AD). The aim of this review is to summarize, within the peripheral immune system, the implications of different types of myeloid cells in AD and VD, with a special focus on post-stroke cognitive impairment and dementia (PSCID). We will review the contributions of the myeloid lineage, from peripheral cells (neutrophils, platelets, monocytes and monocyte-derived macrophages) to central nervous system (CNS)-associated cells (perivascular macrophages and microglia). Finally, we will evaluate different potential strategies for pharmacological modulation of pathological processes mediated by myeloid cell subsets, with an emphasis on neutrophils, their interaction with platelets and the process of immunothrombosis that triggers neutrophil-dependent capillary stall and hypoperfusion, as possible effector mechanisms that may pave the way to novel therapeutic avenues to stop dementia, the epidemic of our time. LINKED ARTICLES: This article is part of a themed issue From Alzheimer's Disease to Vascular Dementia: Different Roads Leading to Cognitive Decline. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.6/issuetoc.

The role of gut microbiota in cerebrovascular disease and related dementia.

In recent years, increasing evidence suggests that commensal microbiota may play an important role not only in health but also in disease including cerebrovascular disease. Gut microbes impact physiology, at least in part, by metabolizing dietary factors and host-derived substrates and then generating active compounds including toxins. The purpose of this current review is to highlight the complex interplay between microbiota, their metabolites. and essential functions for human health, ranging from regulation of the metabolism and the immune system to modulation of brain development and function. We discuss the role of gut dysbiosis in cerebrovascular disease, specifically in acute and chronic stroke phases, and the possible implication of intestinal microbiota in post-stroke cognitive impairment and dementia, and we identify potential therapeutic opportunities of targeting microbiota in this context. LINKED ARTICLES: This article is part of a themed issue From Alzheimer's Disease to Vascular Dementia: Different Roads Leading to Cognitive Decline. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.6/issuetoc.

Ipsilesional Hippocampal GABA Is Elevated and Correlates With Cognitive Impairment and Maladaptive Neurogenesis After Cortical Stroke in Mice.

BACKGROUND: Cognitive dysfunction is a frequent stroke sequela, but its pathogenesis and treatment remain unresolved. Involvement of aberrant hippocampal neurogenesis and maladaptive circuitry remodeling has been proposed, but their mechanisms are unknown. Our aim was to evaluate potential underlying molecular/cellular events implicated. METHODS: Stroke was induced by permanent occlusion of the middle cerebral artery occlusion in 2-month-old C57BL/6 male mice. Hippocampal metabolites/neurotransmitters were analyzed longitudinally by in vivo magnetic resonance spectroscopy. Cognitive function was evaluated with the contextual fear conditioning test. Microglia, astrocytes, neuroblasts, interneurons, γ-aminobutyric acid (GABA), and c-fos were analyzed by immunofluorescence. RESULTS: Approximately 50% of mice exhibited progressive post-middle cerebral artery occlusion cognitive impairment. Notably, immature hippocampal neurons in the impaired group displayed more severe aberrant phenotypes than those from the nonimpaired group. Using magnetic resonance spectroscopy, significant bilateral changes in hippocampal metabolites, such as myo-inositol or N-acetylaspartic acid, were found that correlated, respectively, with numbers of glia and immature neuroblasts in the ischemic group. Importantly, some metabolites were specifically altered in the ipsilateral hippocampus suggesting its involvement in aberrant hippocampal neurogenesis and remodeling processes. Specifically, middle cerebral artery occlusion animals with higher hippocampal GABA levels displayed worse cognitive outcome. Implication of GABA in this setting was supported by the amelioration of ischemia-induced memory deficits and aberrant hippocampal neurogenesis after blocking pharmacologically GABAergic neurotransmission, an intervention which was ineffective when neurogenesis was inhibited. These data suggest that GABA exerts its detrimental effect, at least partly, by affecting morphology and integration of newborn neurons into the hippocampal circuits. CONCLUSIONS: Hippocampal GABAergic neurotransmission could be considered a novel diagnostic and therapeutic target for poststroke cognitive impairment.

Defective hippocampal neurogenesis underlies cognitive impairment by carotid stenosis-induced cerebral hypoperfusion in mice.

Chronic cerebral hypoperfusion due to carotid artery stenosis is a major cause of vascular cognitive impairment and dementia (VCID). Bilateral carotid artery stenosis (BCAS) in rodents is a well-established model of VCID where most studies have focused on white matter pathology and subsequent cognitive deficit. Therefore, our aim was to study the implication of adult hippocampal neurogenesis in hypoperfusion-induced VCID in mice, and its relationship with cognitive hippocampal deficits. Mice were subjected to BCAS; 1 and 3 months later, hippocampal memory and neurogenesis/cell death were assessed, respectively, by the novel object location (NOL) and spontaneous alternation performance (SAP) tests and by immunohistology. Hypoperfusion was assessed by arterial spin labeling-magnetic resonance imaging (ASL-MRI). Hypoperfused mice displayed spatial memory deficits with decreased NOL recognition index. Along with the cognitive deficit, a reduced number of newborn neurons and their aberrant morphology indicated a remarkable impairment of the hippocampal neurogenesis. Both increased cell death in the subgranular zone (SGZ) and reduced neuroblast proliferation rate may account for newborn neurons number reduction. Our data demonstrate quantitative and qualitative impairment of adult hippocampal neurogenesis disturbances associated with cerebral hypoperfusion-cognitive deficits in mice. These findings pave the way for novel diagnostic and therapeutic targets for VCID.

Use of Tenecteplase in Acute Ischemic Stroke in the Time of SARS-CoV-2.

Tenecteplase (TNK) is a fibrinolytic drug that is administrated in a single bolus, recommended in eligible patients with acute ischemic stroke prior to mechanical thrombectomy. This study explores its usefulness in adverse situations, such as the SARS-CoV-2 pandemic. We conducted a retrospective study involving consecutive patients with suspected acute ischemic stroke treated either with intravenous fibrinolysis with alteplase during 2019 or with TNK (.25 mg/kg) between March 2020 and February 2021. A comparative analysis was made to compare patient treatment times and prognosis. A total of 117 patients treated with alteplase and 92 with TNK were included. No significant differences were observed in age, main vascular risk factors or previous treatments. The median National Institutes of Health Stroke Scale was 8 in the alteplase group and 10 in those treated with TNK (P = .13). Combined treatment with mechanical thrombectomy was performed in 47% in the alteplase group and 46.7% in the TNK group; Thrombolysis In Cerebral Infarction scale 2b-3 recanalization was achieved in 83% and 90.7%, respectively (P = .30). There was a decrease in onset-to-needle median time (165 min vs 140 min, P < .01) and no significant variations in door-needle median time. There was no significant difference in the incidence of symptomatic hemorrhagic transformation in mortality or functional independence at 3 months. The easier administration of TNK has improved the accessibility of fibrinolytic therapy, even in adverse circumstances, such as the COVID-19 pandemic. Its use appears to be safe and effective, even in patients who are not candidates for mechanical thrombectomy.

Consensus Recommendations for Standardized Data Elements, Scales, and Time Segmentations in Studies of Human Circadian/Diurnal Biology and Stroke.

Increasing evidence indicates that circadian and diurnal rhythms robustly influence stroke onset, mechanism, progression, recovery, and response to therapy in human patients. Pioneering initial investigations yielded important insights but were often single-center series, used basic imaging approaches, and used conflicting definitions of key data elements, including what constitutes daytime versus nighttime. Contemporary methodologic advances in human neurovascular investigation have the potential to substantially increase understanding, including the use of large multicenter and national data registries, detailed clinical trial data sets, analysis guided by individual patient chronotype, and multimodal computed tomographic and magnetic resonance imaging. To fully harness the power of these approaches to enhance pathophysiologic knowledge, an important foundational step is to develop standardized definitions and coding guides for data collection, permitting rapid aggregation of data acquired in different studies, and ensuring a common framework for analysis. To meet this need, the Leducq Consortium International pour la Recherche Circadienne sur l'AVC (CIRCA) convened a Consensus Statement Working Group of leading international researchers in cerebrovascular and circadian/diurnal biology. Using an iterative, mixed-methods process, the working group developed 79 data standards, including 48 common data elements (23 new and 25 modified/unmodified from existing common data elements), 14 intervals for time-anchored analyses of different granularity, and 7 formal, validated scales. This portfolio of standardized data structures is now available to assist researchers in the design, implementation, aggregation, and interpretation of clinical, imaging, and population research related to the influence of human circadian/diurnal biology upon ischemic and hemorrhagic stroke.

Saturated fatty acid-enriched small extracellular vesicles mediate a crosstalk inducing liver inflammation and hepatocyte insulin resistance.

Background & Aims: Lipotoxicity triggers non-alcoholic fatty liver disease (NAFLD) progression owing to the accumulation of toxic lipids in hepatocytes including saturated fatty acids (SFAs), which activate pro-inflammatory pathways. We investigated the impact of hepatocyte- or circulating-derived small extracellular vesicles (sEV) secreted under NAFLD conditions on liver inflammation and hepatocyte insulin signalling. Methods: sEV released by primary mouse hepatocytes, characterised and analysed by lipidomics, were added to mouse macrophages/Kupffer cells (KC) to monitor internalisation and inflammatory responses. Insulin signalling was analysed in hepatocytes exposed to conditioned media from sEV-loaded macrophages/KC. Mice were i.v. injected sEV to study liver inflammation and insulin signalling. Circulating sEV from mice and humans with NAFLD were used to evaluate macrophage-hepatocyte crosstalk. Results: Numbers of sEV released by hepatocytes increased under NAFLD conditions. Lipotoxic sEV were internalised by macrophages through the endosomal pathway and induced pro-inflammatory responses that were ameliorated by pharmacological inhibition or deletion of Toll-like receptor-4 (TLR4). Hepatocyte insulin signalling was impaired upon treatment with conditioned media from macrophages/KC loaded with lipotoxic sEV. Both hepatocyte-released lipotoxic sEV and the recipient macrophages/KC were enriched in palmitic (C16:0) and stearic (C18:0) SFAs, well-known TLR4 activators. Upon injection, lipotoxic sEV rapidly reached KC, triggering a pro-inflammatory response in the liver monitored by Jun N-terminal kinase (JNK) phosphorylation, NF-κB nuclear translocation, pro-inflammatory cytokine expression, and infiltration of immune cells into the liver parenchyma. sEV-mediated liver inflammation was attenuated by pharmacological inhibition or deletion of TLR4 in myeloid cells. Macrophage inflammation and subsequent hepatocyte insulin resistance were also induced by circulating sEV from mice and humans with NAFLD. Conclusions: We identified hepatocyte-derived sEV as SFA transporters targeting macrophages/KC and activating a TLR4-mediated pro-inflammatory response enough to induce hepatocyte insulin resistance. Impact and Implications: Small extracellular vesicles (sEV) released by the hepatocytes under non-alcoholic fatty liver disease (NAFLD) conditions cause liver inflammation and insulin resistance in hepatocytes via paracrine hepatocyte-macrophage-hepatocyte crosstalk. We identified sEV as transporters of saturated fatty acids (SFAs) and potent lipotoxic inducers of liver inflammation. TLR4 deficiency or its pharmacological inhibition ameliorated liver inflammation induced by hepatocyte-derived lipotoxic sEV. Evidence of this macrophage-hepatocyte interactome was also found in patients with NAFLD, pointing to the relevance of sEV in SFA-mediated lipotoxicity in NAFLD.

Safety and Efficacy of ApTOLL in Patients With Ischemic Stroke Undergoing Endovascular Treatment: A Phase 1/2 Randomized Clinical Trial.

Importance: ApTOLL is a TLR4 antagonist with proven preclinical neuroprotective effect and a safe profile in healthy volunteers. Objective: To assess the safety and efficacy of ApTOLL in combination with endovascular treatment (EVT) for patients with ischemic stroke. Design, Setting, and Participants: This phase 1b/2a, double-blind, randomized, placebo-controlled study was conducted at 15 sites in Spain and France from 2020 to 2022. Participants included patients aged 18 to 90 years who had ischemic stroke due to large vessel occlusion and were seen within 6 hours after stroke onset; other criteria were an Alberta Stroke Program Early CT Score of 6 to 10, estimated infarct core volume on baseline computed tomography perfusion of 5 to 70 mL, and the intention to undergo EVT. During the study period, 4174 patients underwent EVT. Interventions: In phase 1b, 0.025, 0.05, 0.1, or 0.2 mg/kg of ApTOLL or placebo; in phase 2a, 0.05 or 0.2 mg/kg of ApTOLL or placebo; and in both phases, treatment with EVT and intravenous thrombolysis if indicated. Main Outcomes and Measures: The primary end point was the safety of ApTOLL based on death, symptomatic intracranial hemorrhage (sICH), malignant stroke, and recurrent stroke. Secondary efficacy end points included final infarct volume (via MRI at 72 hours), NIHSS score at 72 hours, and disability at 90 days (modified Rankin Scale [mRS] score). Results: In phase Ib, 32 patients were allocated evenly to the 4 dose groups. After phase 1b was completed with no safety concerns, 2 doses were selected for phase 2a; these 119 patients were randomized to receive ApTOLL, 0.05 mg/kg (n = 36); ApTOLL, 0.2 mg/kg (n = 36), or placebo (n = 47) in a 1:1:√2 ratio. The pooled population of 139 patients had a mean (SD) age of 70 (12) years, 81 patients (58%) were male, and 58 (42%) were female. The primary end point occurred in 16 of 55 patients (29%) receiving placebo (10 deaths [18.2%], 4 sICH [7.3%], 4 malignant strokes [7.3%], and 2 recurrent strokes [3.6%]); in 15 of 42 patients (36%) receiving ApTOLL, 0.05 mg/kg (11 deaths [26.2%], 3 sICH [7.2%], 2 malignant strokes [4.8%], and 2 recurrent strokes [4.8%]); and in 6 of 42 patients (14%) receiving ApTOLL, 0.2 mg/kg (2 deaths [4.8%], 2 sICH [4.8%], and 3 recurrent strokes [7.1%]). ApTOLL, 0.2 mg/kg, was associated with lower NIHSS score at 72 hours (mean difference log-transformed vs placebo, -45%; 95% CI, -67% to -10%), smaller final infarct volume (mean difference log-transformed vs placebo, -42%; 95% CI, -66% to 1%), and lower degrees of disability at 90 days (common odds ratio for a better outcome vs placebo, 2.44; 95% CI, 1.76 to 5.00). Conclusions and Relevance: In acute ischemic stroke, 0.2 mg/kg of ApTOLL administered within 6 hours of onset in combination with EVT was safe and associated with a potential meaningful clinical effect, reducing mortality and disability at 90 days compared with placebo. These preliminary findings await confirmation from larger pivotal trials. Trial Registration: ClinicalTrials.gov Identifier: NCT04734548.

Beneficial effect of TLR4 blockade by a specific aptamer antagonist after acute myocardial infarction.

Experimental evidence indicates that the control of the inflammatory response after myocardial infarction is a key strategy to reduce cardiac injury. Cellular damage after blood flow restoration in the heart promotes sterile inflammation through the release of molecules that activate pattern recognition receptors, among which TLR4 is the most prominent. Transient regulation of TLR4 activity has been considered one of the potential therapeutic interventions with greater projection towards the clinic. In this regard, the characterization of an aptamer (4FT) that acts as a selective antagonist for human TLR4 has been investigated in isolated macrophages from different species and in a rat model of cardiac ischemia/reperfusion (I/R). The binding kinetics and biological responses of murine and human macrophages treated with 4FT show great affinity and significant inhibition of TLR4 signaling including the NF-κB pathway and the LPS-dependent increase in the plasma membrane currents (Kv currents). In the rat model of I/R, administration of 4FT following reoxygenation shows amelioration of cardiac injury function and markers, a process that is significantly enhanced when the second dose of 4FT is administered 24 h after reperfusion of the heart. Parameters such as cardiac injury biomarkers, infiltration of circulating inflammatory cells, and the expression of genes associated with the inflammatory onset are significantly reduced. In addition, the expression of anti-inflammatory genes, such as IL-10, and pro-resolution molecules, such as resolvin D1 are enhanced after 4FT administration. These results indicate that targeting TLR4 with 4FT offers new therapeutic opportunities to prevent cardiac dysfunction after infarction.

Tumor Necrosis Factor (TNF)-α-Stimulated Gene 6 (TSG-6): A Promising Immunomodulatory Target in Acute Neurodegenerative Diseases.

Tumor necrosis factor (TNF)-α-stimulated gene 6 (TSG-6), the first soluble chemokine-binding protein to be identified in mammals, inhibits chemotaxis and transendothelial migration of neutrophils and attenuates the inflammatory response of dendritic cells, macrophages, monocytes, and T cells. This immunoregulatory protein is a pivotal mediator of the therapeutic efficacy of mesenchymal stem/stromal cells (MSC) in diverse pathological conditions, including neuroinflammation. However, TSG-6 is also constitutively expressed in some tissues, such as the brain and spinal cord, and is generally upregulated in response to inflammation in monocytes/macrophages, dendritic cells, astrocytes, vascular smooth muscle cells and fibroblasts. Due to its ability to modulate sterile inflammation, TSG-6 exerts protective effects in diverse degenerative and inflammatory diseases, including brain disorders. Emerging evidence provides insights into the potential use of TSG-6 as a peripheral diagnostic and/or prognostic biomarker, especially in the context of ischemic stroke, whereby the pathobiological relevance of this protein has also been demonstrated in patients. Thus, in this review, we will discuss the most recent data on the involvement of TSG-6 in neurodegenerative diseases, particularly focusing on relevant anti-inflammatory and immunomodulatory functions. Furthermore, we will examine evidence suggesting novel therapeutic opportunities that can be afforded by modulating TSG-6-related pathways in neuropathological contexts and, most notably, in stroke.

Comparison of 2 medium cutoff dialyzers versus on-line hemodiafiltration regarding depurative ability and albumin loss: An uncontrolled clinical research.

BACKGROUND: Hemodialysis (HD) techniques that best remove molecules in the middle to high molecular weight range are on-line hemodiafiltration (OL-HDF) and HD with medium cut-off (MCO) membranes. The aim of this study was to compare efficacy and safety of OL-HDF with FxCordiax HDF 800™, with HD with 2 MCO dialyzers: Theranova 500® and the new Elisio 21HX™ dialyzer. METHODS: Fourteen patients following treatment with OL-HDF using FxCordiax HDF 800™ were randomized to receive a consecutive 1-week HD treatment with Theranova 500® and Elisio 21HX™.The reduction rate (RR) of differently sized molecules was compared, as well as the variation rate in molecules smaller than 1000, detected by nuclear magnetic resonance based chemometrics (metabolomics). Albumin loss in dialysate was quantified. RESULTS: Lower RRs were found for molecules around 20 000 with Elisio 21HX™ compared to OL- HDF (RR prolactin 58.5% versus 66.7%, p = 0.034; RR Kappa light chain 63.1% versus 71.8%, p = 0.010). Albumin loss per session was higher with Theranova 500® than with OL-HDF and with Elisio 21HX™ (2249.9 ± 714.1 mg, 815.2 ± 474.0 mg, 442.9 ± 135.9 mg, p < 0.001, respectively). Metabolomic studies suggested, by semi-quantitative analysis, a greater depurative capacity of OL-HDF, followed by Elisio 21HX™, and then Theranova 500®. CONCLUSIONS: In this study, HD with Theranova 500® has proven to be very similar in efficacy to OL-HDF, although with a significantly higher albumin loss. HD with Elisio 21HX™ resulted in lower removal of molecules around 20 000 compared to OL-HDF, with no significant difference compared to Theranova 500®, and with less albumin loss than Theranova 500®.

Endothelial cells regulate astrocyte to neural progenitor cell trans-differentiation in a mouse model of stroke.

The concept of the neurovascular unit emphasizes the importance of cell-cell signaling between neural, glial, and vascular compartments. In neurogenesis, for example, brain endothelial cells play a key role by supplying trophic support to neural progenitors. Here, we describe a surprising phenomenon where brain endothelial cells may release trans-differentiation signals that convert astrocytes into neural progenitor cells in male mice after stroke. After oxygen-glucose deprivation, brain endothelial cells release microvesicles containing pro-neural factor Ascl1 that enter into astrocytes to induce their trans-differentiation into neural progenitors. In mouse models of focal cerebral ischemia, Ascl1 is upregulated in endothelium prior to astrocytic conversion into neural progenitor cells. Injecting brain endothelial-derived microvesicles amplifies the process of astrocyte trans-differentiation. Endothelial-specific overexpression of Ascl1 increases the local conversion of astrocytes into neural progenitors and improves behavioral recovery. Our findings describe an unexpected vascular-regulated mechanism of neuroplasticity that may open up therapeutic opportunities for improving outcomes after stroke.

Diurnal Differences in Immune Response in Brain, Blood and Spleen After Focal Cerebral Ischemia in Mice.

BACKGROUND: The immune response to acute cerebral ischemia is a major factor in stroke pathobiology. Circadian biology modulates some aspects of immune response. The goal of this study is to compare key parameters of immune response during the active/awake phase versus inactive/sleep phase in a mouse model of transient focal cerebral ischemia. METHODS: Mice were housed in normal or reversed light cycle rooms for 3 weeks, and then they were blindly subjected to transient focal cerebral ischemia. Flow cytometry was used to examine immune responses in blood, spleen, and brain at 3 days after ischemic onset. RESULTS: In blood, there were higher levels of circulating T cells in mice subjected to focal ischemia during zeitgeber time (ZT)1-3 (inactive or sleep phase) versus ZT13-15 mice (active or awake phase). In the spleen, organ weight and immune cell numbers were lower in ZT1-3 versus ZT13-15 mice. Consistent with these results, there was an increased infiltration of activated T cells into brain at ZT1-3 compared with ZT13-15. CONCLUSIONS: This proof-of-concept study indicates that there are significant diurnal effects on the immune response after focal cerebral ischemia in mice. Hence, therapeutic strategies focused on immune targets should be reassessed to account for the effects of diurnal rhythms and circadian biology in nocturnal rodent models of stroke.

Fingolimod Does Not Reduce Infarction After Focal Cerebral Ischemia in Mice During Active or Inactive Circadian Phases.

BACKGROUND: It has been reported that the S1P (sphingosine 1-phosphate) receptor modulator fingolimod reduces infarction in rodent models of stroke. Recent studies have suggested that circadian rhythms affect stroke and neuroprotection. Therefore, this study revisited the use of fingolimod in mouse focal cerebral ischemia to test the hypothesis that efficacy might depend on whether experiments were performed during the inactive sleep or active wake phases of the circadian cycle. METHODS: Two different stroke models were implemented in male C57Bl/6 mice-transient middle cerebral artery occlusion and permanent distal middle cerebral artery occlusion. Occlusion occurred either during inactive or active circadian phases. Mice were treated with 1 mg/kg fingolimod at 30- or 60-minute postocclusion and 1 day later for permanent and transient middle cerebral artery occlusion, respectively. Infarct volume, brain swelling, hemorrhagic transformation, and behavioral outcome were assessed at 2 or 3 days poststroke. Three independent experiments were performed in 2 different laboratories. RESULTS: Fingolimod decreased peripheral lymphocyte number in naive mice, as expected. However, it did not significantly affect infarct volume, brain swelling, hemorrhagic transformation, or behavioral outcome at 2 or 3 days after transient or permanent focal cerebral ischemia during inactive or active circadian phases of stroke onset. CONCLUSIONS: Outcomes were not improved by fingolimod in either transient or permanent focal cerebral ischemia during both active and inactive circadian phases. These negative findings suggest that further testing of fingolimod in clinical trials may not be warranted unless translational studies can identify factors associated with fingolimod's efficacy or lack thereof.

The neurovascular unit and systemic biology in stroke - implications for translation and treatment.

Ischaemic stroke is a leading cause of disability and death for which no acute treatments exist beyond recanalization. The development of novel therapies has been repeatedly hindered by translational failures that have changed the way we think about tissue damage after stroke. What was initially a neuron-centric view has been replaced with the concept of the neurovascular unit (NVU), which encompasses neuronal, glial and vascular compartments, and the biphasic nature of neural-glial-vascular signalling. However, it is now clear that the brain is not the private niche it was traditionally thought to be and that the NVU interacts bidirectionally with systemic biology, such as systemic metabolism, the peripheral immune system and the gut microbiota. Furthermore, these interactions are profoundly modified by internal and external factors, such as ageing, temperature and day-night cycles. In this Review, we propose an extension of the concept of the NVU to include its dynamic interactions with systemic biology. We anticipate that this integrated view will lead to the identification of novel mechanisms of stroke pathophysiology, potentially explain previous translational failures, and improve stroke care by identifying new biomarkers of and treatment targets in stroke.

Insulin receptor activation by proinsulin preserves synapses and vision in retinitis pigmentosa.

Synaptic loss, neuronal death, and circuit remodeling are common features of central nervous system neurodegenerative disorders. Retinitis pigmentosa (RP), the leading cause of inherited blindness, is a group of retinal dystrophies characterized by photoreceptor dysfunction and death. The insulin receptor, a key controller of metabolism, also regulates neuronal survival and synaptic formation, maintenance, and activity. Indeed, deficient insulin receptor signaling has been implicated in several brain neurodegenerative pathologies. We present evidence linking impaired insulin receptor signaling with RP. We describe a selective decrease in the levels of the insulin receptor and its downstream effector phospho-S6 in retinal horizontal cell terminals in the rd10 mouse model of RP, as well as aberrant synapses between rod photoreceptors and the postsynaptic terminals of horizontal and bipolar cells. A gene therapy strategy to induce sustained proinsulin, the insulin precursor, production restored retinal insulin receptor signaling, by increasing S6 phosphorylation, without peripheral metabolic consequences. Moreover, proinsulin preserved photoreceptor synaptic connectivity and prolonged visual function in electroretinogram and optomotor tests. These findings point to a disease-modifying role of insulin receptor and support the therapeutic potential of proinsulin in retinitis pigmentosa.

First-in-human phase I clinical trial of a TLR4-binding DNA aptamer, ApTOLL: Safety and pharmacokinetics in healthy volunteers.

ApTOLL is an aptamer that antagonizes Toll-like receptor 4 and improves functional outcomes in models of ischemic stroke and myocardial infarction. The aim of this study was to characterize the safety and pharmacokinetics of ApTOLL in healthy volunteers. A first-in-human dose-ascending, randomized, placebo-controlled phase I clinical trial to assess safety and pharmacokinetics of ApTOLL (30-min infusion intravenously) was performed in 46 healthy adult male volunteers. The study was divided into two parts: part A included seven single ascending dose levels, and part B had one multiple dose cohort. Safety and pharmacokinetic parameters were evaluated. No serious adverse events or biochemistry alterations were detected at any dose nor at any administration pattern studied. Maximum concentration was detected at the end of the infusion and mean half-life was 9.3 h. Interestingly, exposure increased in the first four levels receiving doses from 0.7 mg to 14 mg (AUC of 2,441.26 h∗ng/mL to 23,371.11 h∗ng/mL) but remained stable thereafter (mean of 23,184.61 h∗ng/mL after 70 mg). Consequently, the multiple dose study did not show any accumulation of ApTOLL. These results show an excellent safety and adequate pharmacokinetic profile that, together with the efficacy demonstrated in nonclinical studies, provide the basis to start clinical trials in patients.

Neutrophil Extracellular Trap Targeting Protects Against Ischemic Damage After Fibrin-Rich Thrombotic Stroke Despite Non-Reperfusion.

Stroke is one of the most prevalent diseases worldwide caused primarily by a thrombotic vascular occlusion that leads to cell death. To date, t-PA (tissue-type plasminogen activator) is the only thrombolytic therapy approved which targets fibrin as the main component of ischemic stroke thrombi. However, due to its highly restrictive criteria, t-PA is only administrated to less than 10% of all stroke patients. Furthermore, the research in neuroprotective agents has been extensive with no translational results from medical research to clinical practice up to now. Since we first described the key role of NETs (Neutrophil Extracellular Traps) in platelet-rich thrombosis, we asked, first, whether NETs participate in fibrin-rich thrombosis and, second, if NETs modulation could prevent neurological damage after stroke. To this goal, we have used the thromboembolic in situ stroke model which produces fibrin-rich thrombotic occlusion, and the permanent occlusion of the middle cerebral artery by ligature. Our results demonstrate that NETs do not have a predominant role in fibrin-rich thrombosis and, therefore, DNase-I lacks lytic effects on fibrin-rich thrombosis. Importantly, we have also found that NETs exert a deleterious effect in the acute phase of stroke in a platelet-TLR4 dependent manner and, subsequently, that its pharmacological modulation has a neuroprotective effect. Therefore, our data strongly support that the pharmacological modulation of NETs in the acute phase of stroke, could be a promising strategy to repair the brain damage in ischemic disease, independently of the type of thrombosis involved.

Cav-1 Protein Levels in Serum and Infarcted Brain Correlate with Hemorrhagic Volume in a Mouse Model of Thromboembolic Stroke, Independently of rt-PA Administration.

Thrombolytic therapy with recombinant tissue plasminogen activator (rt-PA) is currently the only FDA-approved drug for acute ischemic stroke. However, its administration is still limited due to the associated increased risk of hemorrhagic transformation (HT). rt-PA may exacerbate blood-brain barrier (BBB) injury by several mechanisms that have not been fully elucidated. Caveolin-1 (Cav-1), a major structural protein of caveolae, has been linked to the endothelial barrier function. The effects of rt-PA on Cav-1 expression remain largely unknown. Here, Cav-1 protein expression after ischemic conditions, with or without rt-PA administration, was analyzed in a murine thromboembolic middle cerebral artery occlusion (MCAO) and in brain microvascular endothelial bEnd.3 cells subjected to oxygen/glucose deprivation (OGD). Our results show that Cav-1 is overexpressed in endothelial cells of infarcted area and in bEnd.3 cell line after ischemia but there is disagreement regarding rt-PA effects on Cav-1 expression between both experimental models. Delayed rt-PA administration significantly reduced Cav-1 total levels from 24 to 72 h after reoxygenation and increased pCav-1/Cav-1 at 72 h in the bEnd.3 cells while it did not modify Cav-1 immunoreactivity in the infarcted area at 24 h post-MCAO. Importantly, tissue Cav-1 positively correlated with Cav-1 serum levels at 24 h post-MCAO and negatively correlated with the volume of hemorrhage after infarction, the latter supporting a protective role of Cav-1 in cerebral ischemia. In addition, the negative association between baseline serum Cav-1 levels and hemorrhagic volume points to a potential usefulness of baseline serum Cav-1 levels to predict hemorrhagic volume, independently of rt-PA administration.

Role of TLR4 in Neutrophil Dynamics and Functions: Contribution to Stroke Pathophysiology.

Background and Purpose: The immune response subsequent to an ischemic stroke is a crucial factor in its physiopathology and outcome. It is known that TLR4 is implicated in brain damage and inflammation after stroke and that TLR4 absence induces neutrophil reprogramming toward a protective phenotype in brain ischemia, but the mechanisms remain unknown. We therefore asked how the lack of TLR4 modifies neutrophil function and their contribution to the inflammatory process. Methods: In order to assess the role of the neutrophilic TLR4 after stroke, mice that do not express TLR4 in myeloid cells (TLR4loxP/Lyz-cre) and its respective controls (TLR4loxP/loxP) were used. Focal cerebral ischemia was induced by occlusion of the middle cerebral artery and infarct size was measured by MRI. A combination of flow cytometry and confocal microscopy was used to assess different neutrophil characteristics (circadian fluctuation, cell surface markers, cell complexity) and functions (apoptosis, microglia engulfment, phagocytosis, NETosis, oxidative burst) in both genotypes. Results: As previously demonstrated, mice with TLR4 lacking-neutrophils had smaller infarct volumes than control mice. Our results show that the absence of TLR4 keeps neutrophils in a steady youth status that is dysregulated, at least in part, after an ischemic insult, preventing neutrophils from their normal circadian fluctuation. TLR4-lacking neutrophils showed a higher phagocytic activity in the basal state, they were preferentially engulfed by the microglia after stroke, and they produced less radical oxygen species (ROS) in the first stage of the inflammatory process. Conclusions: TLR4 is specifically involved in neutrophil dynamics under physiological conditions as well as in stroke-induced tissue damage. This research contributes to the idea that TLR4, especially when targeted in specific cell types, is a potential target for neuroprotective strategies.