Current acute ischemic stroke treatment is limited to reperfusion therapies within the first hours after the onset, underscoring the urgent need for novel therapeutic strategies to mitigate the progression of the brain lesion. Here, we describe the development of bio-inspired synthetic nanoshuttles-lipid nanoparticles (LNPs)-designed to deliver miRNAs and target post-stroke inflammation, a key contributor to secondary brain damage. miRNAs isolated from human induced pluripotent stem cells (hiPSCs) were encapsulated into LNPs prepared by a microfluidic technique. We produced small sized, uniform nanoparticles with high miRNA encapsulation efficiency. LNPs were functionalized with anti-transferrin receptor (TfR) and anti-E-selectin antibodies to target the inflamed cerebral vasculature. We assayed LNPs' effect in a model of vascular inflammation in vitro and in vivo following brain ischemia/reperfusion in mice. In cultured human brain endothelial cells, empty LNPs, dual-targeted empty LNPs, and dual-targeted LNP encapsulating miRNAs (DT-LNP) reduced lipopolysaccharide-induced interleukin-6 production, indicating a modulatory effect of the lipid carrier under our experimental conditions. Mice received, via i.v., either empty LNPs (control) or two different doses of DT-LNPs at reperfusion following brain ischemia. The low dose of DT-LNPs reduced infarct volume and tended to improve the neurological deficit, and to downregulate the expression of IL-1β and E-selectin mRNAs, whereas the high dose did not. Altogether, these findings provide proof-of-concept that TfR-E-selectin-targeted LNPs encapsulating hiPSC-derived miRNAs represent a promising therapeutic approach for stroke.

Recent studies suggest that blood–brain barrier (BBB) disruption plays a key role in the clinical course and bleeding risk of brain arteriovenous malformations (bAVMs). The tight junctions (TJs) are complex endothelial transmembrane proteins with a significant physical contribution to BBB disruption. In this study, we hypothesized that bAVMs display a different TJ pattern than other vascular malformations and normal brain tissue. We studied the expression of claudin-5 and occludin as essential factors for functional TJs. Human specimens of surgically resected cavernomas (CCMs) (n = 9), bAVMs (n = 17), and perilesional brain parenchyma (6 from CCMs and 16 from bAVM patients) were analyzed via immunofluorescence staining, transmission electron microscopy (TEM), and Western blot tests. Compared to perilesional parenchyma, bAVMs showed a significant decrease in TJ protein expression, and these alterations were more apparent in ruptured bAVMs than in unruptured bAVMs or CCMs. TEM images provided evidence of disrupted connectivity between endothelial cells of bAVMs. This is the first clinical investigation that studies the expression of TJs in human bAVMs and their surrounding parenchyma. Despite the limitations of the sample size, we found significant differences in the expression and composition of TJs in bAVMs when compared to surrounding parenchyma and other vascular lesions such as CCMs. These results add further evidence to the role of BBB disruption in the clinical course of bAVM. A deeper understanding of these mechanisms may lead to the development of new therapeutic targets and management strategies for bAVMs.

Background Severe brain ischemia is associated with life‐threatening edema and inflammation. Interleukin‐1 is a crucial mediator of inflammation, and its blockade showed benefits in experimental stroke. We studied anakinra, a modified recombinant human interleukin‐1 receptor antagonist, in mouse models of moderate to severe ischemia/reperfusion and large hemispheric infarctions. Due to anakinra's short half‐life, we used a novel subcutaneous infusion protocol and tested 2 drug doses. Methods and Results We performed transient or permanent intraluminal middle cerebral artery occlusion (MCAo) in male C57BL/6J and Balb/c mice, the latter of which have poorer collaterals. Mice received a subcutaneous anakinra bolus (24 mg/kg), followed by continuous infusion of either 24 or 120 mg/kg per day, starting at reperfusion or 15 minutes after permanent MCAo. We evaluated acute (24 hours/48 hours) infarct volume and edema by magnetic resonance imaging, neurological function, and inflammatory responses. The mortality rate tended to be higher in Balb/c compared with C57BL/6J mice. In both strains, prolonged ischemia expanded the infarct size, with intraluminal permanent MCAo resulting in larger hemispheric infarctions and edema than transient MCAo. The high dose of anakinra reduced infarct volume and inflammation in C57BL/6 mice and improved the functional deficits in Balb/c mice following transient MCAo. It also showed a trend toward reducing infarction and edema after permanent MCAo in C57BL/6 mice. Conclusions The study demonstrates that a high dose of anakinra improves outcomes in mouse models of moderate infarction following ischemia/reperfusion, whereas its effect was less pronounced in a malignant hemispheric infarction model without reperfusion, where only a nonsignificant trend toward protection was observed.

The 14-3-3 protein family plays a crucial role in cellular signaling and neuronal survival. Although 14-3-3 isoforms have been involved in the etiology of some neurodegenerative disorders, their role remains poorly understood in alpha-synucleinopathies. In this study, we wanted to characterize 14-3-3 gene expression patterns in the brain and periphery of patients with Lewy body pathology (LBP). We analyzed messenger RNA (mRNA) expression of all seven 14-3-3 isotypes in the temporal cortex and caudate nucleus of brains with pure and common LBP, as well as in cerebrospinal fluid, peripheral blood, and platelets. Stratifin (SFN) protein expression was assessed by immunohistochemistry and enzyme-linked immunosorbent assay. Effects of SFN overexpression on astrocyte activation were investigated in vitro. In the temporal cortex of LBP patients, six out of seven 14-3-3 genes were significantly downregulated, while SFN was markedly up-regulated. SFN protein localized to neurofibrillary tangles in the parahippocampal region and reactive astrocytes in white matter. In vitro, SFN treatment induced biphasic changes in astrocytic glial fibrillary acidic protein expression, suggesting a role in glial activation. Peripheral analysis revealed no significant 14-3-3 mRNA changes in blood, but platelet expression patterns inversely correlated with brain tissue; six isoforms were up-regulated, and SFN was strongly downregulated in dementia with Lewy bodies (DLB) platelets. Our findings revealed an important shift in 14-3-3 isotype expression in the temporal cortex of LBP patients, where SFN up-regulation reflected its colocalization with tau pathology and astrocytic reactivity. The inverse expression profile in platelets in DLB highlights their potential as accessible biomarkers for molecular changes occurring in the brain.

BACKGROUND:Spontaneous aneurysmal subarachnoid hemorrhage induces early blood-brain barrier permeability dysfunction, although its clinical relevance and underlying mechanisms remain poorly understood. We aimed to evaluate the association between blood-brain barrier disruption, quantified with dynamic contrast-enhanced magnetic resonance imaging at the end of the early brain injury period, circulating neuroinflammatory mediators, and long-term clinical outcomes. METHODS:We analyzed a prospective cohort of subarachnoid hemorrhage patients who underwent dynamic contrast-enhanced magnetic resonance imaging at a median (interquartile range) of 4 (2-6) days after clinical onset. Permeability maps were used to obtain K-trans values as a measure of increased blood-brain barrier permeability in the whole brain, gray matter, and white matter. Circulating neuroinflammatory molecules, including IL (interleukin) 8 and PDGF (platelet-derived growth factor), were measured using Multiplex-ELISA in blood samples collected concurrently with magnetic resonance imaging acquisition. Poor clinical outcome was defined as a modified Rankin Scale score of >2 at 90 days. Associations between K-trans values, neuroinflammatory mediators, and clinical outcomes were assessed using univariate and multivariate regression models. RESULTS:From 153 patients initially screened, 96 were finally included (63% females; median age, 55 years; 43% premorbid hypertension; 32% World Federation of Neurosurgical Societies grade 4-5; 31% poor outcome). In adjusted linear regression analyses, higher K-trans values were significantly associated with increased IL-8 (P=0.001) and PDGF (P=0.018) levels. In univariate analysis, K-trans values in white matter were significantly higher in patients with poor clinical outcome (median [interquartile range], 2.5 [2.07-6.09] ×10-3·min-1) compared with good clinical outcome (median [interquartile range], 2.0 [1.60-2.42] ×10-3·min-1; P<0.001). In models adjusted by age, World Federation of Neurosurgical Societies, hypertension, intraparenchymal hematoma, aneurysm size, and time to magnetic resonance imaging, elevated K-trans values remained independently associated with poor outcome (adjusted odds ratio per interquartile range increase, 3.31 [95% CI, 1.485-7.377]; P=0.003). CONCLUSIONS:Increased blood-brain barrier permeability correlates with circulating neuroinflammatory mediator levels and is associated with poor clinical recovery at 3 months. These findings support the potential role of white matter permeability alterations as both biomarker and therapeutic target in subarachnoid hemorrhage.

BACKGROUND:Past failures in translating stroke cerebroprotection provoked calls for a more rigorous methodological approach, leading to the stroke preclinical assessment network SPAN (Stroke Preclinical Assessment Network), where uric acid (UA) treatment exceeded a prespecified efficacy boundary for the primary functional outcome. Still, successful translation to humans requires confirmation of the effect of UA across key biological variables relevant to patients with stroke. METHODS:We measured the effects of intravenous UA treatment (16 mg/kg) versus intravenous saline in groups of animals enrolled in the SPAN network with diverse comorbidities, sex, and age. The masked study drug or placebo was administered during reperfusion in rodents undergoing a transient middle cerebral artery filament occlusion. The primary outcome was the modified corner test index at day 30 poststroke, and numerous secondary outcomes were collected. A modified intention-to-treat population was used in the analysis. We tested for any interactions with sex, age, and comorbidities (obesity-induced hyperglycemia and hypertension). RESULTS:In total, 710 animals were randomized to receive either intravenous UA or saline. After accounting for procedural dropouts and exclusions from treatment, a total of 687 animals were qualified and analyzed, including 458 assigned to UA and 229 to intravenous saline control. UA-treated animals exhibited a better primary functional outcome at day 30 (probability, 0.56 [95% CI, 0.52-0.60]; P=0.006). UA-treated animals also had a better corner test index at day 7 (probability, 0.55 [95% CI, 0.5-0.59]; P=0.035) and a higher survival rate at day 30 (hazard ratio, 1.41 [95% CI, 1.08-1.83]; P=0.011). Brain morphometry at day 2 and 30 was comparable between the treatment groups. The improved functional outcome and survival in UA-treated animals were preserved across different species, sexes, ages, and comorbidities. CONCLUSIONS:UA provides ischemic stroke cerebroprotection across key relevant biological variables, making it a promising intervention to be further tested in human clinical trials.

Monomeric C-reactive protein (mCRP) is a pro-inflammatory molecule generated by the dissociation of native CRP. Clinical and experimental studies suggest that mCRP deposition in the brain induces Alzheimer's disease (AD) pathology and cognitive loss. Pathological neuroinflammation is increasingly suggested as relevant in AD. Innovative therapies against neuroinflammation are desperately needed, and inhibitors of the enzyme soluble epoxide hydrolase (sEH) are a promising new generation of anti-inflammatory drugs. Mouse primary microglia and BV2 cell line cultures were exposed to mCRP to analyze its pro-inflammatory mechanisms. sEH inhibitors, both newly synthesized UB-SCG-55 and UB-SCG-65, and the reference agent TPPU, were tested for their anti-inflammatory action against mCRP. Phenotypic changes were analyzed through cell imaging techniques, as well as molecular analysis of inflammatory mediators and gene activation pathways. Results show that mCRP triggers a pro-inflammatory response through three main inflammatory pathways: iNOS, NLRP3, and COX-2, followed by increased cytokine generation. Polarization of microglia toward a M1-like phenotype was confirmed by morphological analysis. Also, mCRP can bind to and cross the cell membrane, providing further insight into its mechanisms of action. sEH inhibitors were effective against mCRP induction of a reactive microglial phenotype. The first-line compound UB-SCG-55 emerged as the most potent anti-inflammatory against mCRP injury. Therefore, the direct activation of microglia by mCRP provides evidence of its role in triggering and exacerbating neurodegenerative diseases with a neuroinflammatory component, such as AD. Furthermore, the protection given by inhibitors of sEH confirms its potential as innovative drugs against deleterious effects of neuroinflammation.

Through GWAS studies we identified PATJ associated with functional outcome after ischemic stroke (IS). The aim of this study was to determine PATJ role in brain endothelial cells (ECs) in the context of stroke outcome. PATJ expression analyses in patient’s blood revealed that: (i) the risk allele of rs76221407 induces higher expression of PATJ, (ii) PATJ is downregulated 24 h after IS, and (iii) its expression is significantly lower in those patients with functional independence, measured at 3 months with the modified Rankin scale ((mRS) ≤2), compared to those patients with marked disability (mRS = 4–5). In mice brains, PATJ was also downregulated in the injured hemisphere at 48 h after ischemia. Oxygen-glucose deprivation and hypoxia-dependent of Hypoxia Inducible Factor-1α also caused PATJ depletion in ECs. To study the effects of PATJ downregulation, we generated PATJ-knockdown human microvascular ECs. Their transcriptomic profile evidenced a complex cell reprogramming involving Notch, TGF-ß, PI3K/Akt, and Hippo signaling that translates in morphological and functional changes compatible with endothelial to mesenchymal transition (EndMT). PATJ depletion caused loss of cell-cell adhesion, upregulation of metalloproteases, actin cytoskeleton remodeling, cytoplasmic accumulation of the signal transducer C-terminal transmembrane Mucin 1 (MUC1-C) and downregulation of Notch and Hippo signaling. The EndMT phenotype of PATJ-depleted cells was associated with the nuclear recruitment of MUC1-C, YAP/TAZ, β-catenin, and ZEB1. Our results suggest that PATJ downregulation 24 h after IS promotes EndMT, an initial step prior to secondary activation of a pro-angiogenic program. This effect is associated with functional independence suggesting that activation of EndMT shortly after stroke onset is beneficial for stroke recovery.

Histone deacetylase SIRT1 represses gene expression through the deacetylation of histones and transcription factors and is involved in the protective cell response to stress and aging. However, upon endoplasmic reticulum (ER) stress, SIRT1 impairs the IRE1α branch of the unfolded protein response (UPR) through the inhibition of the transcriptional activity of XBP-1 and SIRT1 deficiency is beneficial under these conditions. We hypothesized that SIRT1 deficiency may unlock the blockade of transcription factors unrelated to the UPR promoting the synthesis of chaperones and improving the stability of immature proteins or triggering the clearance of unfolded proteins. SIRT1+/+ and SIRT1−/− fibroblasts were exposed to the ER stress inducer tunicamycin and cell survival and expression of heat shock proteins were analyzed 24 h after the treatment. We observed that SIRT1 loss significantly reduced cell sensitivity to ER stress and showed that SIRT1−/− but not SIRT1+/+ cells constitutively expressed high levels of phospho-STAT3 and heat shock proteins. Hsp70 silencing in SIRT1−/− cells abolished the resistance to ER stress. Furthermore, accumulation of ubiquitinated proteins was lower in SIRT1−/− than in SIRT1+/+ cells. Our data showed that SIRT1 deficiency enabled chaperones upregulation and boosted the proteasome activity, two processes that are beneficial for coping with ER stress.

Hyperglycemia has been linked to worsening outcomes after subarachnoid hemorrhage (SAH). Nevertheless, the mechanisms involved in the pathogenesis of SAH have been scarcely evaluated so far. The role of hyperglycemia was assessed in an experimental model of SAH by T2 weighted, dynamic contrast-enhanced magnetic resonance imaging (T2W and DCE-MRI), [18F]BR-351 PET imaging and immunohistochemistry. Measures included the volume of bleeding, the extent of cerebral infarction and brain edema, blood brain barrier disruption (BBBd), neutrophil infiltration and matrix metalloprotease (MMP) activation. The neurofunctional outcome, neurodegeneration and myelinization were also investigated. The induction of hyperglycemia increased mortality, the size of the ischemic lesion, brain edema, neurodegeneration and worsened neurological outcome during the first 3 days after SAH in rats. In addition, these results show for the first time the exacerbating effect of hyperglycemia on in vivo MMP activation, Intercellular Adhesion Molecule 1 (ICAM-1) expression and neutrophil infiltration together with increased BBBd, bleeding volume and fibrinogen accumulation at days 1 and 3 after SAH. Notably, these data provide valuable insight into the detrimental effect of hyperglycemia on early BBB damage mediated by neutrophil infiltration and MMP activation that could explain the worse prognosis in SAH.

Microglia play key roles in the post-ischemic inflammatory response and damaged tissue removal reacting rapidly to the disturbances caused by ischemia and working to restore the lost homeostasis. However, the modified environment, encompassing ionic imbalances, disruption of crucial neuron-microglia interactions, spreading depolarization, and generation of danger signals from necrotic neurons, induce morphological and phenotypic shifts in microglia. This leads them to adopt a proinflammatory profile and heighten their phagocytic activity. From day three post-ischemia, macrophages infiltrate the necrotic core while microglia amass at the periphery. Further, inflammation prompts a metabolic shift favoring glycolysis, the pentose-phosphate shunt, and lipid synthesis. These shifts, combined with phagocytic lipid intake, drive lipid droplet biogenesis, fuel anabolism, and enable microglia proliferation. Proliferating microglia release trophic factors contributing to protection and repair. However, some microglia accumulate lipids persistently and transform into dysfunctional and potentially harmful foam cells. Studies also showed microglia that either display impaired apoptotic cell clearance, or eliminate synapses, viable neurons, or endothelial cells. Yet, it will be essential to elucidate the viability of engulfed cells, the features of the local environment, the extent of tissue damage, and the temporal sequence. Ischemia provides a rich variety of region- and injury-dependent stimuli for microglia, evolving with time and generating distinct microglia phenotypes including those exhibiting proinflammatory or dysfunctional traits and others showing pro-repair features. Accurate profiling of microglia phenotypes, alongside with a more precise understanding of the associated post-ischemic tissue conditions, is a necessary step to serve as the potential foundation for focused interventions in human stroke.

Diseases of the central nervous system (CNS) are often associated with vascular disturbances or inflammation and frequently both. Consequently, endothelial cells and macrophages are key cellular players that mediate pathology in many CNS diseases. Macrophages in the brain consist of the CNS-associated macrophages (CAMs) [also referred to as border-associated macrophages (BAMs)] and microglia, both of which are close neighbours or even form direct contacts with endothelial cells in microvessels. Recent progress has revealed that different macrophage populations in the CNS and a subset of brain endothelial cells are derived from the same erythromyeloid progenitor cells. Macrophages and endothelial cells share several common features in their life cycle—from invasion into the CNS early during embryonic development and proliferation in the CNS, to their demise. In adults, microglia and CAMs have been implicated in regulating the patency and diameter of vessels, blood flow, the tightness of the blood–brain barrier, the removal of vascular calcification, and the life-time of brain endothelial cells. Conversely, CNS endothelial cells may affect the polarization and activation state of myeloid populations. The molecular mechanisms governing the pas de deux of brain macrophages and endothelial cells are beginning to be deciphered and will be reviewed here.

We aimed to analyse whether patients with ischaemic stroke (IS) occurring within eight days after the onset of COVID-19 (IS-COV) are associated with a specific aetiology of IS. We used SUPERGNOVA to identify genome regions that correlate between the IS-COV cohort (73 IS-COV cases vs. 701 population controls) and different aetiological subtypes. Polygenic risk scores (PRSs) for each subtype were generated and tested in the IS-COV cohort using PRSice-2 and PLINK to find genetic associations. Both analyses used the IS-COV cohort and GWAS from MEGASTROKE (67,162 stroke patients vs. 454,450 population controls), GIGASTROKE (110,182 vs. 1,503,898), and the NINDS Stroke Genetics Network (16,851 vs. 32,473). Three genomic regions were associated (p-value < 0.05) with large artery atherosclerosis (LAA) and cardioembolic stroke (CES). We found four loci targeting the genes PITX2 (rs10033464, IS-COV beta = 0.04, p-value = 2.3 × 10−2, se = 0.02), previously associated with CES, HS6ST1 (rs4662630, IS-COV beta = −0.04, p-value = 1.3 × 10−3, se = 0.01), TMEM132E (rs12941838 IS-COV beta = 0.05, p-value = 3.6 × 10−4, se = 0.01), and RFFL (rs797989 IS-COV beta = 0.03, p-value = 1.0 × 10−2, se = 0.01). A statistically significant PRS was observed for LAA. Our results suggest that IS-COV cases are genetically similar to LAA and CES subtypes. Larger cohorts are needed to assess if the genetic factors in IS-COV cases are shared with the general population or specific to viral infection.

BACKGROUND: Respiratory and urinary tract infections are frequent complications in patients with severe stroke. Stroke-associated infection is mainly due to opportunistic commensal bacteria of the microbiota that may translocate from the gut. We investigated the mechanisms underlying gut dysbiosis and poststroke infection. METHODS: Using a model of transient cerebral ischemia in mice, we explored the relationship between immunometabolic dysregulation, gut barrier dysfunction, gut microbial alterations, and bacterial colonization of organs, and we explored the effect of several drug treatments. RESULTS: Stroke-induced lymphocytopenia and widespread colonization of lung and other organs by opportunistic commensal bacteria. This effect correlated with reduced gut epithelial barrier resistance, and a proinflammatory sway in the gut illustrated by complement and nuclear factor-κB activation, reduced number of gut regulatory T cells, and a shift of gut lymphocytes to γδT cells and T helper 1/T helper 17 phenotypes. Stroke increased conjugated bile acids in the liver but decreased bile acids and short-chain fatty acids in the gut. Gut fermenting anaerobic bacteria decreased while opportunistic facultative anaerobes, notably Enterobacteriaceae, suffered an expansion. Anti-inflammatory treatment with a nuclear factor-κB inhibitor fully abrogated the Enterobacteriaceae overgrowth in the gut microbiota induced by stroke, whereas inhibitors of the neural or humoral arms of the stress response were ineffective at the doses used in this study. Conversely, the anti-inflammatory treatment did not prevent poststroke lung colonization by Enterobacteriaceae. CONCLUSIONS: Stroke perturbs homeostatic neuro-immuno-metabolic networks facilitating a bloom of opportunistic commensals in the gut microbiota. However, this bacterial expansion in the gut does not mediate poststroke infection.

Uric acid (UA) is a strong endogenous antioxidant that neutralizes the toxicity of peroxynitrite and other reactive species on the neurovascular unit generated during and after acute brain ischemia. The realization that a rapid reduction of UA levels during an acute ischemic stroke was associated with a worse stroke outcome paved the way to investigate the value of exogenous UA supplementation to counteract the progression of redox-mediated ischemic brain damage. The long translational journey for UA supplementation recently reached a critical milestone when the results of the multicenter NIH stroke preclinical assessment network (SPAN) were reported. In a novel preclinical paradigm, 6 treatment candidates including UA supplementation were selected and tested in 6 independent laboratories following predefined criteria and strict methodological rigor. UA supplementation was the only intervention in SPAN that exceeded the prespecified efficacy boundary with male and female animals, young mice, young rats, aging mice, obese mice, and spontaneously hypertensive rats. This unprecedented achievement will allow UA to undergo clinical testing in a pivotal clinical trial through a NIH StrokeNet thrombectomy endovascular platform created to assess new treatment strategies in patients treated with mechanical thrombectomy. UA is a particularly appealing adjuvant intervention for mechanical thrombectomy because it targets the microcirculatory hypoperfusion and oxidative stress that limits the efficacy of this therapy. This descriptive review aims to summarize the translational development of UA supplementation, highlighting those aspects that likely contributed to its success. It includes having a well-defined target and mechanism of action, and an approach that simultaneously integrated rigorous preclinical assessment, with epidemiologic and preliminary human intervention studies. Validation of the clinical value of UA supplementation in a pivotal trial would confirm the translational value of the SPAN paradigm in preclinical research.

Stroke stands as a major cause of death or chronic disability globally. Nevertheless, existing optimal treatments are limited to reperfusion therapies during the acute phase of ischemic stroke. To gain insights into stroke physiopathology and develop innovative therapeutic approaches, in vivo rodent models of stroke play a fundamental role. The availability of genetically modified animals has particularly propelled the use of mice as experimental stroke models. In stroke patients, occlusion of the middle cerebral artery (MCA) is a common occurrence. Consequently, the most prevalent experimental model involves intraluminal occlusion of the MCA, a minimally invasive technique that doesn't require craniectomy. This procedure involves inserting a monofilament through the external carotid artery (ECA) and advancing it through the internal carotid artery (ICA) until it reaches the branching point of the MCA. After a 45 min arterial occlusion, the monofilament is removed to allow reperfusion. Throughout the process, cerebral blood flow is monitored to confirm the reduction during occlusion and subsequent recovery upon reperfusion. Neurological and tissue outcomes are evaluated using behavioral tests and magnetic resonance imaging (MRI) studies.

Vascular endothelial function is challenged during cerebral ischemia and reperfusion. The endothelial responses are involved in inflammatory leukocyte attraction, adhesion and infiltration, blood-brain barrier leakage, and angiogenesis. This study investigated gene expression changes in brain endothelial cells after acute ischemic stroke using transcriptomics and translatomics. We isolated brain endothelial mRNA by: (i) translating ribosome affinity purification, enabling immunoprecipitation of brain endothelial ribosome-attached mRNA for translatome sequencing and (ii) isolating CD31+ endothelial cells by fluorescence-activating cell sorting for classical transcriptomic analysis. Both techniques revealed similar pathways regulated by ischemia but they showed specific differences in some transcripts derived from non-endothelial cells. We defined a gene set characterizing the endothelial response to acute stroke (24h) by selecting the differentially expressed genes common to both techniques, thus corresponding with the translatome and minimizing non-endothelial mRNA contamination. Enriched pathways were related to inflammation and immunoregulation, angiogenesis, extracellular matrix, oxidative stress, and lipid trafficking and storage. We validated, by flow cytometry and immunofluorescence, the protein expression of several genes encoding cell surface proteins. The inflammatory response was associated with the endothelial upregulation of genes related to lipid storage functions and we identified lipid droplet biogenesis in the endothelial cells after ischemia. The study reports a robust translatomic signature of brain endothelial cells after acute stroke and identifies enrichment in novel pathways involved in membrane signaling and lipid storage. Altogether these results highlight the endothelial contribution to the inflammatory response, and identify novel molecules that could be targets to improve vascular function after ischemic stroke.