MicroRNAs, contained in exosomes or freely circulating in the plasma, might play a pivotal role in the infarct-sparing effect exerted by remote limb ischemic postconditioning (RLIP). The aims of the present study were: (1) To evaluate the effect of pure exosomes isolated from plasma of animals subjected to RLIP systemically administered to ischemic rats; (2) To finely dissect exosomes content in terms of miRNAs; (3) To select those regulatory miRNAs specifically expressed in protective exosomes and to identify molecular pathways involved in their neurobeneficial effects. Circulating exosomes were isolated from blood of animals exposed to RLIP and administered to animals exposed to tMCAO by intracerebroventricular, intraperitoneal or intranasal routes. Exosomal miRNA signature was evaluated by microarray and FISH analysis. Plasmatic exosomes isolated from plasma of RLIP rats attenuated cerebral ischemia reperfusion injury and improved neurological functions until 3 days after ischemia induction. Interestingly, miR-702-3p and miR-423-5p seem to be mainly involved in exosome protective action by modulating NOD1 and NLRP3, two key triggers of neuroinflammation and neuronal death. Collectively, the results of the present work demonstrated that plasma-released exosomes after RLIP may transfer a neuroprotective signal to the brain of ischemic animals, thus representing a potentially translatable therapeutic strategy in stroke.

OBJECTIVE: Ischemic complications account for significant patient morbidity following aneurysmal subarachnoid hemorrhage (aSAH). The Prevention and Treatment of Vasospasm with Clazosentan (REACT) study was designed to assess the safety and efficacy of clazosentan, an endothelin receptor antagonist, in preventing clinical deterioration due to delayed cerebral ischemia (DCI) in patients with aSAH.
METHODS: REACT was a prospective, multicenter, randomized, double-blind, phase 3 study. Eligible patients had aSAH secured by surgical clipping or endovascular coiling, and had presented with thick and diffuse clot on admission CT scan. Patients were randomized (1:1 ratio) to 15 mg/hour intravenous clazosentan or placebo within 96 hours of the aSAH for up to 14 days, in addition to standard of care treatment including oral or intravenous nimodipine. The primary efficacy endpoint was the occurrence of clinical deterioration due to DCI up to 14 days after initiation of the study drug. The main secondary endpoint was the occurrence of clinically relevant cerebral infarction at day 16 after study drug initiation. Other secondary endpoints included clinical outcome assessed on the modified Rankin Scale (mRS) and the Glasgow Outcome Scale-Extended (GOSE) at week 12 post-aSAH. Imaging and clinical endpoints were centrally adjudicated.
RESULTS: A total of 409 patients were randomized between February 2019 and May 2022 across 74 international sites. Three patients did not start study treatment and were not included in the analysis set. The occurrence of clinical deterioration due to DCI was 15.8% (32/202 patients) in the clazosentan group and 17.2% (35/204 patients) in the placebo group, and the difference was not statistically significant (relative risk reduction [RRR] 7.2%, 95% CI -42.6% to 39.6%, p = 0.734). A nonsignificant RRR of 34.1% (95% CI -21.3% to 64.2%, p = 0.177) was observed in clinically relevant cerebral infarcts treated with clazosentan (7.4%, 15/202) versus placebo (11.3%, 23/204). Rescue therapy was less frequently needed for patients treated with clazosentan compared to placebo (10.4%, 21/202 vs 18.1%, 37/204; RRR 42.6%, 95% CI 5.4%-65.2%). A nonsignificant relative risk increase of 25.4% (95% CI -10.7% to 76.0%, p = 0.198) was reported in the risk of poor GOSE and mRS scores with clazosentan (24.8%, 50/202) versus placebo (20.1%, 41/204) at week 12 post-aSAH. Treatment-emergent adverse events were similar to those reported previously.
CONCLUSIONS: Clazosentan administered for up to 14 days at 15 mg/hour had no significant effect on the occurrence of clinical deterioration due to DCI. Clinical trial registration no.: NCT03585270 (ClinicalTrials.gov) EU clinical trial registration no.: 2018-000241-39 (clinicaltrialsregister.eu).

Aim: Although lactate supplementation at the reperfusion stage of ischemic stroke has been shown to offer neuroprotection, whether the role of accumulated lactate at the ischemia phase is neuroprotection or not remains largely unknown. Thus, in this study, we aimed to investigate the roles and mechanisms of accumulated brain lactate at the ischemia stage in regulating brain injury of ischemic stroke. Methods and Results: Pharmacological inhibition of lactate production by either inhibiting LDHA or glycolysis markedly attenuated the mouse brain injury of ischemic stroke. In contrast, additional lactate supplement further aggravates brain injury, which may be closely related to the induction of neuronal death and A1 astrocytes. The contributing roles of increased lactate at the ischemic stage may be related to the promotive formation of protein lysine lactylation (Kla), while the post-treatment of lactate at the reperfusion stage did not influence the brain protein Kla levels with neuroprotection. Increased protein Kla levels were found mainly in neurons by the HPLC-MS/MS analysis and immunofluorescent staining. Then, pharmacological inhibition of lactate production or blocking the lactate shuttle to neurons showed markedly decreased protein Kla levels in the ischemic brains. Additionally, Ldha specific knockout in astrocytes (Aldh1l1 CreERT2; Ldha fl/fl mice, cKO) mice with MCAO were constructed and the results showed that the protein Kla level was decreased accompanied by a decrease in the volume of cerebral infarction in cKO mice compared to the control groups. Furthermore, blocking the protein Kla formation by inhibiting the writer p300 with its antagonist A-485 significantly alleviates neuronal death and glial activation of cerebral ischemia with a reduction in the protein Kla level, resulting in extending reperfusion window and improving functional recovery for ischemic stroke. Conclusion: Collectively, increased brain lactate derived from astrocytes aggravates ischemic brain injury by promoting the protein Kla formation, suggesting that inhibiting lactate production or the formation of protein Kla at the ischemia stage presents new therapeutic targets for the treatment of ischemic stroke.

Although ischemia increases the abundance of plasminogen activator inhibitor-1 (PAI-1), its source and role in the ischemic brain remain unclear. We detected PAI-1-immunoreactive cells with morphological features of reactive astrocytes in the peri-ischemic cortex of mice after an experimentally-induced ischemic lesion, and of a chimpanzee that suffered a naturally-occurring stroke. We found that although the abundance of PAI-1 increases 24 hours after the onset of the ischemic injury in a non-reperfusion murine model of ischemic stroke, at that time-point there is no difference in astrocytic reactivity and the volume of the ischemic lesion between wild-type (Wt) animals and in mice either genetically deficient (PAI-1-/-) or overexpressing PAI-1 (PAI-1Tg). In contrast, 72 hours later astrocytic reactivity and the volume of the ischemic lesion were decreased in PAI-1-/- mice and increased in PAI-1Tg animals. Our immunoblottings and fractal analysis studies show that the abundance of astrocytic PAI-1 rises during the recovery phase from a hypoxic injury, which in turn increases the abundance of glial fibrillary acidic protein (GFAP) and triggers morphological features of reactive astrocytes. These studies indicate that cerebral ischemia-induced release of astrocytic PAI-1 triggers astrocytic reactivity associated with enlargement of the necrotic core.

UNASSIGNED: Carotid siphon calcification (CSC) serves as a marker of atherosclerosis and therefore may influence the outcome after subarachnoid hemorrhage (aSAH). We aimed to analyze the impact of CSC on neurological outcomes, ischemia, and vasospasm.
UNASSIGNED: A total of 716 patients with aSAH were treated between December 2004 and June 2016 in our central European tertiary neurovascular care center in Essen, Germany. CSC was recorded using the Woodcock scale (grades 0-3) on a computed tomography scan. Study end points included an unfavorable outcome at 6 months post-aSAH (modified Rankin Scale score ≥4), vasospasm, and early cerebral ischemia (72 hours) and delayed cerebral ischemia (delayed cerebral ischemia; >72 hours) in the follow-up computed tomography scans. The associations were adjusted for patients\' baseline characteristics and secondary complications. Finally, within a subgroup analysis, patients with and without daily aspirin intake after endovascular aneurysm occlusion were compared.
UNASSIGNED: Increasing grades of CSC were associated with lower rates of vasospasm in the anterior circulation. Severe CSC (grade 3) was independently related to the risk of an unfavorable outcome (adjusted odds ratio [aOR], 4.06 [95% CI, 1.98-8.33]; P<0.001) and early cerebral ischemia (aOR, 1.58 [95% CI, 1.03-2.43]; P=0.035) but not delayed cerebral ischemia (aOR, 1.08 [95% CI, 0.67-1.73]; P=0.763). In the aspirin subgroup analysis, the negative effect of severe CSC on functional outcome remained significant only in aSAH cases without aspirin (aOR, 5.47 [95% CI, 2.38-12.54]; P<0.001). In contrast, there was no association between severe CSC and unfavorable outcomes among individuals with daily aspirin intake (aOR, 0.84 [95% CI, 0.59-4.21]; P=0.603).
UNASSIGNED: Our data suggest CSC as a cerebrovascular risk factor resulting in higher rates of early cerebral ischemia and unfavorable outcomes after aSAH. However, by increasing arterial stiffness, CSC might lower the probability of vasospasm, which could explain the missing link between CSC and delayed cerebral ischemia. Additionally, aspirin intake seems to potentially mitigate the negative impact of CSC on aSAH outcome. Further investigations are needed to confirm the observations from the present study.

BACKGROUND: Long-term disability after stroke is standardly assessed 3 months post-onset, using the modified Rankin Scale (mRS). The value of an early, day 4 mRS assessment for projecting the 3-month disability outcome has not been formally investigated.
METHODS: In this cohort of patients with acute cerebral ischemia and intracranial hemorrhage, we analyzed day 4 and day 90 mRS assessments in the NIH Field Administration of Stroke Therapy- Magnesium (FAST-MAG) Phase 3 trial. The performance of day 4 mRS, alone and as part of multivariate models, in predicting day 90 mRS was assessed using correlation coefficients, percent agreement, and the kappa statistics.
RESULTS: Among the 1573 acute cerebrovascular disease (ACVD) patients, 1206 (76.7%) had acute cerebral ischemia (ACI), while 367 (23.3%) had intracranial hemorrhage. Among all 1573 ACVD patients, day 4 mRS and day 90 mRS correlated strongly, Spearman\'s rho=0.79, in unadjusted analysis with weighted kappa of 0.59. For dichotomized outcomes, simple carry-forward of the day 4 mRS performed fairly well in agreeing with day 90 mRS: mRS 0-1 (kappa=0.67), 85.4%; mRS 0-2 (k=0.59), 79.5%; fatal outcome, 88% (k=0.33). Correlations of 4d and 90d mRS were stronger for ACI than ICH patients, 0.76 vs 0.71.
CONCLUSIONS: In this acute cerebrovascular disease patient cohort, assessment of global disability performed on day 4 is highly informative regarding long-term, 3-month mRS disability outcome, alone, and even more strongly in combination with baseline prognostic variables. The day 4 mRS is a useful measure for imputing the final patient disability outcome in clinical trials and quality improvement programs.

Results of previous studies suggested that programmed cell death 4 (PDCD4) was overexpressed in cerebral ischemia (CI), and mothers against decapentaplegic homolog 1 (SMAD1) is a transcription factor of PDCD4, and it is also elevated in CI; however, the regulatory mechanism of SMAD1/PDCD4 axis in CI remains unclear. The current work has been designed to explore the role and associated mechanisms of SMAD1/PDCD4 in CI. PDCD4 and SMAD1 expressions have been examined by real-time reverse transcription-polymerase chain reaction (RT-qPCR) method, and receiver operating characteristic (ROC) curve analysis has been performed to determine the potential diagnostic value of PDCD4 and SMAD1. An oxygen-glucose deprivation (OGD) model has been used to investigate the effects of PDCD4 and SMAD1 on CI in vitro. Cell apoptosis was evaluated using TdT-mediated dUTP nick end labeling (TUNEL) assays. The interaction between SMAD1 and PDCD4 axis has been confirmed by using dual-luciferase reporter as well as chromatin immunoprecipitation (Ch-IP) assays. Finally, the effects of SMAD1/PDCD4 axis on the ferroptosis of neuron cells have been examined. PDCD4 was overexpressed in blood samples of CI patients. ROC analysis showed the AUC for PDCD4 was 0.7478, and NIHSS and MRS scores were positively correlated with PDCD4 expression. Moreover, the cellular OGD model was established and knockdown of PDCD4 suppressed the apoptosis of neurons. Besides, knockdown of PDCD4 also inhibited ferroptosis of OGD-treated neuron cells in vitro. Additionally, SMAD1 was upregulated in blood samples of CI patients, NIHSS and MRS scores were positively correlated with SMAD1 expression, and SMAD1 is a transcriptional factor of PDCD4, and SMAD1 could transcriptionally regulate the expression of PDCD4. Finally, SMAD1 could regulate the ferroptosis of neuron cells through regulating the transcription of PDCD4. The SMAD1/PDCD4 axis regulates the growth, apoptosis, and ferroptosis of neuron cells, suggesting that targeting the SMAD1/PDCD4 axis may be a potential therapeutic method.

UNASSIGNED: Ischemic stroke (IS) is one of the leading causes of death and disability in the world, and alcohol consumption has been gaining attention as an independent risk factor for IS. Blood-brain barrier (BBB) dysfunction and neuroinflammation are the core of cerebral ischemia/reperfusion (I/R) injury, and pericytes play a crucial role in the structure and function. This study is to explore the effects of long-term alcohol consumption on IS and the potential mechanisms of pericytes.
UNASSIGNED: Rat models of long-term alcohol intake followed by transient middle cerebral artery occlusion stroke (EtOH+tMCAO) and cell models of oxygen-glucose deprivation/reoxygenation (OGD/R) with alcohol pre-treatment were constructed.
UNASSIGNED: Worsened infarct volume, neurological scores, and BBB disruption were observed in the EtOH+tMCAO group compared with the tMCAO group, and immunofluorescence staining showed increased pericytes NLPR3 inflammasome activation at the ischemic penumbra. In vitro, pericyte mortality and LDH release elevated pre-treated by alcohol after OGD/R, and amplified expression of NLRP3 inflammasome was detected by Western blotting and qPCR. Alcohol pre-treatment activated the TLR4/NF-κB pathway, and transfecting pericytes with TLR4-small interfering RNA (siRNA) to block TLR4 signaling markedly restrained NLRP3 inflammasome over-activation. Injecting TAK-242 in rats alleviated neurological impairment caused by alcohol.
UNASSIGNED: Long-term alcohol pre-treatment aggravated ischemic stroke-induced brain damage by activating NLRP3 inflammasome via TLR4/NF-κB signaling pathway in the pericytes.

The present study investigated the neuroprotective properties of whole plants of Grewia bilamellata Gagnep. extract (GBEE) against cerebral ischemia by harnessing both In vivo studies in a rat model and In silico studies focusing on nitric oxide synthase (NOS) inhibition. High-resolution liquid chromatography‒mass spectrometry (HR LC‒MS) analysis identified 32 phytochemicals in the GBEE, 15 of which adhered to Lipinski\'s rule of five. These compounds exhibited diverse physicochemical properties and high binding affinity to NOS, with cleomiscosin D showing the greatest potential. In vivo, GBEE had significant neuroprotective effects on bilateral common carotid artery occlusion/reperfusion (BCCAO/R) in rats, especially at doses of 200 mg/kg and 400 mg/kg body weight. GBEE treatment improved brain function, as evidenced by EEG normalization, substantial reductions in cerebral infarction size, mitigated neuronal loss, and the restoration of regular histological arrangement in the CA1 hippocampal area of the brain. Furthermore, GBEE enhanced antioxidant defenses by augmenting the activity of catalase (CAT) and superoxide dismutase (SOD), reducing malondialdehyde (MDA) levels, and restoring reduced glutathione (GSH) levels. These effects were accompanied by a decrease in nitric oxide (NO) levels, indicative of attenuated oxidative and nitrosative stress. Collectively, our findings suggest that GBEE is a promising natural therapeutic agent that may prevent or alleviate ischemic brain injury through a multifaceted mechanism involving NOS inhibition and attenuation of the oxidative stress response. This study highlights the therapeutic potential of GBEE and warrants further research into its mechanism of action and possible clinical applications.

The absence of blood flow in cerebral ischemic conditions triggers a multitude of intricate pathophysiological mechanisms, including excitotoxicity, oxidative stress, neuroinflammation, disruption of the blood-brain barrier and white matter disarrangement. Despite numerous experimental studies conducted in preclinical settings, existing treatments for cerebral ischemia (CI), such as mechanical and pharmacological therapies, remain constrained and often entail significant side effects. Therefore, there is an imperative to explore innovative strategies for addressing CI outcomes. Cannabidiol (CBD), the most abundant non-psychotomimetic compound derived from Cannabis sativa, is a pleiotropic substance that interacts with diverse molecular targets and has the potential to influence various pathophysiological processes, thereby contributing to enhanced outcomes in CI. This chapter provides a comprehensive overview of the primary effects of CBD in in vitro and diverse animal models of CI and delves into some of its plausible mechanisms of neuroprotection.