Neuroprotection for Stroke: Current Status and Future Perspectives

Neuroprotection aims to prevent salvageable neurons from dying. Despite showing efficacy in experimental stroke studies, the concept of neuroprotection has failed in clinical trials. Reasons for the translational difficulties include a lack of methodological agreement between preclinical and clinical studies and the heterogeneity of stroke in humans compared to homogeneous strokes in animal models. Even when the international recommendations for preclinical stroke research, the Stroke Academic Industry Roundtable (STAIR) criteria, were followed, we have still seen limited success in the clinic, examples being NXY-059 and haematopoietic growth factors which fulfilled nearly all the STAIR criteria. However, there are a number of neuroprotective treatments under investigation in clinical trials such as hypothermia and ebselen. Moreover, promising neuroprotective treatments based on a deeper understanding of the complex pathophysiology of ischemic stroke such as inhibitors of NADPH oxidases and PSD-95 are currently evaluated in preclinical studies. Further concepts to improve translation include the investigation of neuroprotectants in multicenter preclinical Phase III-type studies, improved animal models, and close alignment between clinical trial and preclinical methodologies. Future successful translation will require both new concepts for preclinical testing and innovative approaches based on mechanistic insights into the ischemic cascade.     

Circulating MicroRNA Profiling Identifies Distinct MicroRNA Signatures in Acute Ischemic Stroke and Transient Ischemic Attack Patients

Transient ischemic attack (TIA) refers to a momentary neurologic deficit caused by focal cerebral, spinal or retinal ischemic insult. TIA is associated with a high risk of impending acute ischemic stroke (AIS), a neurologic dysfunction characterized by focal cerebral, spinal or retinal infarction. Understanding the differences in molecular pathways in AIS and TIA has merit for deciphering the underlying cause for neuronal deficits with long-term effects and high risks of morbidity and mortality. In this study, we performed comprehensive investigations into the circulating microRNA (miRNA) profiles of AIS (n = 191) and TIA (n = 61) patients. We performed RNA-Seq on serum samples collected within 24 hrs of clinical diagnosis and randomly divided the study populations into discovery and validation cohorts. We identified a panel of 11 differentially regulated miRNAs at FDR < 0.05. Hsa-miR-548c-5p, -20a-5p, -18a-5p, -484, -652-3p, -486-3p, -24-3p, -181a-5p and -222-3p were upregulated, while hsa-miR-500a-3p and -206 were downregulated in AIS patients compared to TIA patients. We also probed the previously validated gene targets of our identified miRNA panel to highlight the molecular pathways affected in AIS. Moreover, we developed a multivariate classifier with potential utilization as a discriminative biomarker for AIS and TIA patients. The underlying molecular pathways in AIS compared to TIA may be explored further in functional studies for therapeutic targeting in clinical translation.     

Heparin and Arginine Based Plasmin Nanoformulation for Ischemic Stroke Therapy

Ischemic stroke is the most common type of stroke and thrombolytic therapy is the only approved treatment. However, current thrombolytic therapy with tissue plasminogen activator (tPA) is often hampered by the increased risk of hemorrhage. Plasmin, a direct fibrinolytic, has a significantly superior hemostatic safety profile; however, if injected intravenously it becomes rapidly inactivated by anti-plasmin. Nanoformulations have been shown to increase drug stability and half-life and hence could be applied to increase the plasmin therapeutic efficacy. Here in this paper, we report a novel heparin and arginine-based plasmin nanoformulation that exhibits increased plasmin stability and efficacy. In vitro studies revealed significant plasmin stability in the presence of anti-plasmin and efficient fibrinolytic activity. In addition, these particles showed no significant toxicity or oxidative stress effects in human brain microvascular endothelial cells, and no significant blood brain barrier permeability. Further, in a mouse photothrombotic stroke model, plasmin nanoparticles exhibited significant efficacy in reducing stroke volume without overt intracerebral hemorrhage (ICH) compared to free plasmin treatment. The study shows the potential of a plasmin nanoformulation in ischemic stroke therapy.     

CCL5 Levels Predict Stroke Volume Growth in Acute Ischemic Stroke and Significantly Diminish in Hemorrhagic Stroke Patients

Stroke remains an important health challenge. Here, we study whether circulating chemokine (C-C motif) ligand 5 (CCL5) levels may predict clinical outcomes for stroke patients. A total of 100 consecutive stroke patients (36 acute ischemic and 64 hemorrhagic) were admitted to the stroke unit. Clinical history data and monitoring parameters were recorded. Blood serum was collected at days 0, 1, and hospital discharge to measure CCL5 levels by ELISA. Infarct or hemorrhagic volume, neurological severity (NIHSS), and functional prognosis (mRankin scale) were measured as clinical outcomes. CCL5 levels were lower in patients with hemorrhagic stroke than in patients with acute ischemic stroke. No differences were found between females and males in both types of stroke. Ischemic stroke patients whose infarct volume grew had lower CCL5 levels at day 0. Levels of CCL5 in ischemic and hemorrhagic patients were not associated with more severe symptoms/worse prognosis (NIHSS > 3; mRankin > 2) at admission or at 3 months. CCL5 could be used as a diagnostic marker to distinguish between ischemic and hemorrhagic strokes. Furthermore, CCL5 levels could predict the infarct volume outcomes in ischemic patients.     

Fatty Acid-Binding Proteins: Their Roles in Ischemic Stroke and Potential as Drug Targets

Stroke is among the leading causes of death and disability worldwide. However, despite long-term research yielding numerous candidate neuroprotective drugs, there remains a lack of effective neuroprotective therapies for ischemic stroke patients. Among the factors contributing to this deficiency could be that single-target therapy is insufficient in addressing the complex and extensive mechanistic basis of ischemic brain injury. In this context, lipids serve as an essential component of multiple biological processes and play important roles in the pathogenesis of numerous common neurological diseases. Moreover, in recent years, fatty acid-binding proteins (FABPs), a family of lipid chaperone proteins, have been discovered to be involved in the onset or development of several neurodegenerative diseases, including Alzheimer’s and Parkinson’s disease. However, comparatively little attention has focused on the roles played by FABPs in ischemic stroke. We have recently demonstrated that neural tissue-associated FABPs are involved in the pathological mechanism of ischemic brain injury in mice. Here, we review the literature published in the past decade that has reported on the associations between FABPs and ischemia and summarize the relevant regulatory mechanisms of FABPs implicated in ischemic injury. We also propose candidate FABPs that could serve as potential therapeutic targets for ischemic stroke.     

Brain Energy Metabolism in Ischemic Stroke: Effects of Smoking and Diabetes

Proper regulation of energy metabolism in the brain is crucial for maintaining brain activity in physiological and different pathophysiological conditions. Ischemic stroke has a complex pathophysiology which includes perturbations in the brain energy metabolism processes which can contribute to worsening of brain injury and stroke outcome. Smoking and diabetes are common risk factors and comorbid conditions for ischemic stroke which have also been associated with disruptions in brain energy metabolism. Simultaneous presence of these conditions may further alter energy metabolism in the brain leading to a poor clinical prognosis after an ischemic stroke event. In this review, we discuss the possible effects of smoking and/or diabetes on brain glucose utilization and mitochondrial energy metabolism which, when present concurrently, may exacerbate energy metabolism in the ischemic brain. More research is needed to investigate brain glucose utilization and mitochondrial oxidative metabolism in ischemic stroke in the presence of smoking and/or diabetes, which would provide further insights on the pathophysiology of these comorbid conditions and facilitate the development of therapeutic interventions.     

Therapeutic Effect of Rapamycin on TDP-43-Related Pathogenesis in Ischemic Stroke

Stroke is a major cause of death and disability across the world, and its detrimental impact should not be underestimated. Therapies are available and effective for ischemic stroke (e.g., thrombolytic recanalization and mechanical thrombectomy); however, there are limitations to therapeutic interventions. Recanalization therapy has developed dramatically, while the use of adjunct neuroprotective agents as complementary therapies remains deficient. Pathological TAR DNA-binding protein (TDP-43) has been identified as a major component of insoluble aggregates in numerous neurodegenerative pathologies, including ALS, FTLD and Alzheimer’s disease. Here, we show that increased pathological TDP-43 fractions accompanied by impaired mitochondrial function and increased gliosis were observed in an ischemic stroke rat model, suggesting a pathological role of TDP-43 in ischemic stroke. In ischemic rats administered rapamycin, the insoluble TDP-43 fraction was significantly decreased in the ischemic cortex region, accompanied by a recovery of mitochondrial function, the attenuation of cellular apoptosis, a reduction in infarct areas and improvements in motor defects. Accordingly, our results suggest that rapamycin provides neuroprotective benefits not only by ameliorating pathological TDP-43 levels, but also by reversing mitochondrial function and attenuating cell apoptosis in ischemic stroke.     

Identification of Novel Circulating miRNAs in Patients with Acute Ischemic Stroke

Ischemic strokes are associated with significant morbidity and mortality, but currently there are no reliable prognostic or diagnostic blood biomarkers. MicroRNAs (miRNAs) regulate various molecular pathways and may be used as biomarkers. Using RNA-Seq, we conducted comprehensive circulating miRNA profiling in patients with ischemic stroke compared with healthy controls. Samples were collected within 24 h of clinical diagnosis. Stringent analysis criteria of discovery (46 cases and 95 controls) and validation (47 cases and 96 controls) cohorts led to the identification of 10 differentially regulated miRNAs, including 5 novel miRNAs, with potential diagnostic significance. Hsa-miR-451a was the most significantly upregulated miRNA (FC; 4.8, FDR; 3.78 × 10⁻⁸⁵), while downregulated miRNAs included hsa-miR-574-5p and hsa-miR-142-3p, among others. Importantly, we computed a multivariate classifier based on the identified miRNA panel to differentiate between ischemic stroke patients and healthy controls, which showed remarkably high sensitivity (0.94) and specificity (0.99). The area under the ROC curve was 0.97 and it is superior to other current available biomarkers. Moreover, in samples collected one month following stroke, we found sustained upregulation of hsa-miR-451a and downregulation of another 5 miRNAs. Lastly, we report 3 miRNAs that were significantly associated with poor clinical outcomes of stroke, as defined by the modified Rankin scores. The clinical translation of the identified miRNA panel may be explored further.     

急性缺血性脑卒中静脉溶栓临床观察与探讨

目的评价急性缺血性脑卒中发病3h内行静脉溶栓的疗效及安全性。方法将61例急性缺血性脑卒中患者分为溶栓组31例,对照组30例,予以低分子肝素。按改良爱丁堡斯堪的那维亚评分标准(CSS),分别于治疗前及治疗后6h、24h、3d、7d及14d记录患者神经功能缺损,并于治疗后1个月及3个月随访患者日常生活能力(ADL量表)。结果溶栓治疗组神经功能改善明显优于对照组(P<0.01),出血并发症无明显增加(P>0.05)。结论 Rt-PA用于急性缺血性脑卒中超早期(3h)治疗疗效肯定,安全性相对较高。     

疏血通注射液治疗急性缺血性脑中风疗效

目的评价疏血通注射液治疗脑梗死(ACI)的有效性。方法选择脑梗死60例,随机分为治疗组和对照组各30例,患者采用疏血通和丹参注射液对照治疗并进行对比临床观察。结果表明疏血通治疗脑缺血疗效明显(P<0.01)对血粘度、脑血循环均有明显改变。在治疗前后对患者进行定期的临床神经功能缺损评分(欧洲卒中评分量表-ESS)、日常生活活动能力(ADL)调查。结果21d后治疗组和对照组差异有显著性(P<0.001);90d后治疗组和对照组ADL有效率分别为差异有显著性(P<0.05);结论疏血通注射液治疗脑梗死更有效。     

Fatty Acids and Lipid Paradox-Neuroprotective Biomarkers in Ischemic Stroke

Stroke is the primary cause of death and disability worldwide, with ischemic stroke up to 80% of the total cases. Lipid profile was established as a major risk factor for stroke, but recent studies show a paradoxical relationship between serum values and the outcome of stroke patients. Our study aims to analyze the impact of the classic extended lipid profile, including fatty acids as potential neuroprotective biomarkers for the outcome of ischemic stroke patients. We included 298 patients and collected clinical, paraclinical, and outcome parameters. We used a method consisting of high-performance liquid chromatography coupled with mass spectrometry to quantify serum fatty acids. We observed a negative correlation between National Institutes of Health Stroke Scale (NIHSS) at admission and total cholesterol (p = 0.040; r = −0.120), respectively triglycerides (p = 0.041; r = −0.122). The eicosapentaenoic to arachidonic acid ratio has a negative correlation, while the docosahexaenoic to eicosapentaenoic acid ratio positively correlates with all the prognostic parameters, showing a potential neuroprotective role for eicosapentaenoic acid in preventing severe ischemic stroke. The impact of the lipid profile paradox and the dependency relationship with the fatty acids represent a significant predictive factor for the functional and disability prognostic of ischemic stroke patients.     

The Role of Matrix Metalloproteinase Polymorphisms in Ischemic Stroke

Stroke remains the fifth leading cause of mortality in the United States with an annual rate of over 128,000 deaths per year. Differences in incidence, pathogenesis, and clinical outcome have long been noted when comparing ischemic stroke among different ethnicities. The observation that racial disparities exist in clinical outcomes after stroke has resulted in genetic studies focusing on specific polymorphisms. Some studies have focused on matrix metalloproteinases (MMPs). MMPs are a ubiquitous group of proteins with extensive roles that include extracellular matrix remodeling and blood-brain barrier disruption. MMPs play an important role in ischemic stroke pathophysiology and clinical outcome. This review will evaluate the evidence for associations between polymorphisms in MMP-1, 2, 3, 9, and 12 with ischemic stroke incidence, pathophysiology, and clinical outcome. The role of polymorphisms in MMP genes may influence the presentation of ischemic stroke and be influenced by racial and ethnic background. However, contradictory evidence for the role of MMP polymorphisms does exist in the literature, and further studies will be necessary to consolidate our understanding of these multi-faceted proteins.     

Melatonin-Based Therapeutics for Neuroprotection in Stroke

The present review paper supports the approach to deliver melatonin and to target melatonin receptors for neuroprotection in stroke. We discuss laboratory evidence demonstrating neuroprotective effects of exogenous melatonin treatment and transplantation of melatonin-secreting cells in stroke. In addition, we describe a novel mechanism of action underlying the therapeutic benefits of stem cell therapy in stroke, implicating the role of melatonin receptors. As we envision the clinical entry of melatonin-based therapeutics, we discuss translational experiments that warrant consideration to reveal an optimal melatonin treatment strategy that is safe and effective for human application.     

ERK1/2 Activity Is Critical for the Outcome of Ischemic Stroke

Ischemic disorders are the leading cause of death worldwide. The extracellular signal-regulated kinases 1 and 2 (ERK1/2) are thought to affect the outcome of ischemic stroke. However, it is under debate whether activation or inhibition of ERK1/2 is beneficial. In this study, we report that the ubiquitous overexpression of wild-type ERK2 in mice (ERK2^wt) is detrimental after transient occlusion of the middle cerebral artery (tMCAO), as it led to a massive increase in infarct volume and neurological deficits by increasing blood–brain barrier (BBB) leakiness, inflammation, and the number of apoptotic neurons. To compare ERK1/2 activation and inhibition side-by-side, we also used mice with ubiquitous overexpression of the Raf-kinase inhibitor protein (RKIP^wt) and its phosphorylation-deficient mutant RKIP^S153A, known inhibitors of the ERK1/2 signaling cascade. RKIP^wt and RKIP^S153A attenuated ischemia-induced damages, in particular via anti-inflammatory signaling. Taken together, our data suggest that stimulation of the Raf/MEK/ERK1/2-cascade is severely detrimental and its inhibition is rather protective. Thus, a tight control of the ERK1/2 signaling is essential for the outcome in response to ischemic stroke.     

Leonurine Reduces Oxidative Stress and Provides Neuroprotection against Ischemic Injury via Modulating Oxidative and NO/NOS Pathway

Leonurine (Leo) has been found to have neuroprotective effects against cerebral ischemic injury. However, the exact molecular mechanism underlying its neuroprotective ability remains unclear. The aim of the present study was to investigate whether Leo could provide protection through the nitric oxide (NO)/nitric oxide synthase (NOS) pathway. We firstly explored the effects of NO/NOS signaling on oxidative stress and apoptosis in in vivo and in vitro models of cerebral ischemia. Further, we evaluated the protective effects of Leo against oxygen and glucose deprivation (OGD)-induced oxidative stress and apoptosis in PC12 cells. We found that the rats showed anxiety-like behavior, and the morphology and number of neurons were changed in a model of photochemically induced cerebral ischemia. Both in vivo and in vitro results show that the activity of superoxide dismutase (SOD) and glutathione (GSH) contents were decreased after ischemia, and reactive oxygen species (ROS) and malondialdehyde (MDA) levels were increased, indicating that cerebral ischemia induced oxidative stress and neuronal damage. Moreover, the contents of NO, total NOS, constitutive NOS (cNOS) and inducible NOS (iNOS) were increased after ischemia in rat and PC12 cells. Treatment with L-nitroarginine methyl ester (L-NAME), a nonselective NOS inhibitor, could reverse the change in NO/NOS expression and abolish these detrimental effects of ischemia. Leo treatment decreased ROS and MDA levels and increased the activity of SOD and GSH contents in PC12 cells exposed to OGD. Furthermore, Leo reduced NO/NOS production and cell apoptosis, decreased Bax expression and increased Bcl-2 levels in OGD-treated PC12 cells. All the data suggest that Leo protected against oxidative stress and neuronal apoptosis in cerebral ischemia by inhibiting the NO/NOS system. Our findings indicate that Leo could be a potential agent for the intervention of ischemic stroke and highlighted the NO/NOS-mediated oxidative stress signaling.     

Circulating MicroRNAs and Extracellular Vesicle-Derived MicroRNAs as Predictors of Functional Recovery in Ischemic Stroke Patients: A Systematic Review and Meta-Analysis

Stroke accounts for the second leading cause of death and a major cause of disability, with limited therapeutic strategy in both the acute and chronic phases. Blood-based biomarkers are intensively researched and widely recognized as useful tools to predict the prognoses of patients confronted with therapeutically limited diseases. We performed a systematic review of the circulating biomarkers in IS patients with prognostic value, with a focus on microRNAs and exosomes as predictive biomarkers of motor and cognitive recovery. We identified 63 studies, totalizing 72 circulating biomarkers with prognostic value in stroke recovery, as follows: 68 miRNAs and exosomal-miRNAs being identified as predictive for motor recovery after stroke, and seven biomarkers being predictive for cognitive recovery. Twelve meta-analyses were performed using effect sizes (random-effects and fixed-effects model). The most significant correlation findings obtained after pooling were with miR-21, miR-29b, miR-125b-5p, miR-126, and miR-335. We identified several miRNAs that were correlated with clinical outcomes of stroke severity and recovery after ischemic stroke, providing predictive information on motor and cognitive recovery. Based on the current state of research, we identified serum miR-9 and neutrophil miR-29b as the most promising biomarkers for in-depth follow-up studies, followed by serum miR-124 and plasma miR-125b.     

Antiplatelet Usage Impacts Clot Density in Acute Anterior Circulation Ischemic Stroke

We explored whether clot density in middle cerebral artery (MCA) occlusion is related to clinical variables, stroke etiology, blood constituents, and prestroke medication. We performed a retrospective chart review of patients with acute ischemic stroke of the anterior circulation admitted to two Central European stroke centers. The acquisition of non-contrast enhanced CT (NECT) and CT angiography (CTA) within 4.5 h of symptom onset was obligatory. We assessed the site of MCA occlusion as well as density, area, and length of the clot in 150 patients. The Hounsfield unit values for the clot were divided with contralateral MCA segment to yield relative Hounsfield Unit ratio (rHU). The site of the vessel occlusion (M1 vs. M2) and antiplatelet usage, but not stroke etiology, significantly influenced rHU. We found an inverse correlation of rHU with erythrocyte count (p < 0.001). The multivariate analysis revealed that a higher rHU (i.e., clot being more hyperdense) was more likely with the use of antiplatelets (OR 4.24, CI 1.10–16.31, p = 0.036). Erythrocyte (OR 0.18, CI 0.05–0.55, p = 0.003), and thrombocyte counts (OR 0.99, CI 0.98–0.99, p = 0.029) were associated with odds for more hypodense clots (lower rHU). Our study disclosed that antiplatelet therapy impacts the composition of intracranial clots of the anterior circulation.     

Glibenclamide for the Treatment of Ischemic and Hemorrhagic Stroke

Ischemic and hemorrhagic strokes are associated with severe functional disability and high mortality. Except for recombinant tissue plasminogen activator, therapies targeting the underlying pathophysiology of central nervous system (CNS) ischemia and hemorrhage are strikingly lacking. Sur1-regulated channels play essential roles in necrotic cell death and cerebral edema following ischemic insults, and in neuroinflammation after hemorrhagic injuries. Inhibiting endothelial, neuronal, astrocytic and oligodendroglial sulfonylurea receptor 1–transient receptor potential melastatin 4 (Sur1–Trpm4) channels and, in some cases, microglial K_ATP (Sur1–Kir6.2) channels, with glibenclamide is protective in a variety of contexts. Robust preclinical studies have shown that glibenclamide and other sulfonylurea agents reduce infarct volumes, edema and hemorrhagic conversion, and improve outcomes in rodent models of ischemic stroke. Retrospective studies suggest that diabetic patients on sulfonylurea drugs at stroke presentation fare better if they continue on drug. Additional laboratory investigations have implicated Sur1 in the pathophysiology of hemorrhagic CNS insults. In clinically relevant models of subarachnoid hemorrhage, glibenclamide reduces adverse neuroinflammatory and behavioral outcomes. Here, we provide an overview of the preclinical studies of glibenclamide therapy for CNS ischemia and hemorrhage, discuss the available data from clinical investigations, and conclude with promising preclinical results that suggest glibenclamide may be an effective therapeutic option for ischemic and hemorrhagic stroke.     

Chemical Conditioning as an Approach to Ischemic Stroke Tolerance: Mitochondria as the Target

It is well established that the brain can be prepared to resist or tolerate ischemic stroke injury, and mitochondrion is a major target for this tolerance. The preparation of ischemic stroke tolerance can be achieved by three major approaches: ischemic conditioning, hypoxic conditioning and chemical conditioning. In each conditioning approach, there are often two strategies that can be used to achieve the conditioning effects, namely preconditioning (Pre-C) and postconditioning (Post-C). In this review, we focus on chemical conditioning of mitochondrial proteins as targets for neuroprotection against ischemic stroke injury. Mitochondrial targets covered include complexes I, II, IV, the ATP-sensitive potassium channel (mitoKATP), adenine dinucleotide translocase (ANT) and the mitochondrial permeability transition pore (mPTP). While numerous mitochondrial proteins have not been evaluated in the context of chemical conditioning and ischemic stroke tolerance, the paradigms and approaches reviewed in this article should provide general guidelines on testing those mitochondrial components that have not been investigated. A deep understanding of mitochondria as the target of chemical conditioning for ischemic stroke tolerance should provide valuable insights into strategies for fighting ischemic stroke, a leading cause of death in the world.