Astrocytes in functional recovery following central nervous system injuries.

Astrocytes are increasingly recognised as partaking in complex homeostatic mechanisms critical for regulating neuronal plasticity following central nervous system (CNS) insults. Ischaemic stroke and traumatic brain injury are associated with high rates of disability and mortality. Depending on the context and type of injury, reactive astrocytes respond with diverse morphological, proliferative and functional changes collectively known as astrogliosis, which results in both pathogenic and protective effects. There is a large body of research on the negative consequences of astrogliosis following brain injuries. There is also growing interest in how astrogliosis might in some contexts be protective and help to limit the spread of the injury. However, little is known about how astrocytes contribute to the chronic functional recovery phase following traumatic and ischaemic brain insults. In this review, we explore the protective functions of astrocytes in various aspects of secondary brain injury such as oedema, inflammation and blood-brain barrier dysfunction. We also discuss the current knowledge on astrocyte contribution to tissue regeneration, including angiogenesis, neurogenesis, synaptogenesis, dendrogenesis and axogenesis. Finally, we discuss diverse astrocyte-related factors that, if selectively targeted, could form the basis of astrocyte-targeted therapeutic strategies to better address currently untreatable CNS disorders.

MiR-182 Inhibition Protects Against Experimental Stroke in vivo and Mitigates Astrocyte Injury and Inflammation in vitro via Modulation of Cortactin Activity.

Ischemic stroke remains a devastating cerebrovascular disease that accounts for a high proportion of mortality and disability worldwide. MicroRNAs (miRNAs) are a class of small non-coding RNAs that are responsible for regulation of post-transcriptional gene expression, and growing evidence supports a role for miRNAs in stroke injury and recovery. The current study examined the role of miR-182 in experimental stroke using both in vitro and in vivo models of ischemic injury. Brain levels of miR-182 significantly increased after transient middle cerebral artery occlusion (MCAO) in mice and in primary astrocyte cultures subjected to combined oxygen-glucose deprivation/reperfusion (OGD/R) injury. In vivo, stroke volume and neurological score were significantly improved by pre-treatment with miR-182 antagomir. Astrocyte cultures stressed with OGD/R resulted in mitochondrial fragmentation and downregulation of cortactin, an actin-binding protein. Inhibition of miR-182 significantly preserved cortactin expression, reduced mitochondrial fragmentation and improved astrocyte survival after OGD/R. In parallel, lipopolysaccharide (LPS)-induced nitric-oxide release in astrocyte cultures was significantly reduced by miR-182 inhibition, translating to reduced injury in primary neuronal cultures subjected to conditioned medium from LPS-treated astrocytes. These findings identify miR-182 and/or cortactin as potential clinical targets to preserve mitochondrial structure and mitigate neuroinflammation and cell death after ischemic stroke.

Synthesis and Development of a Novel First-in-Class Cofilin Inhibitor for Neuroinflammation in Hemorrhagic Brain Injury.

Intracerebral hemorrhage (ICH) is devastating among stroke types with high mortality. To date, not a single therapeutic intervention has been successful. Cofilin plays a critical role in inflammation and cell death. In the current study, we embarked on designing and synthesizing a first-in-class small-molecule inhibitor of cofilin to target secondary complications of ICH, mainly neuroinflammation. A series of compounds were synthesized, and two lead compounds SZ-3 and SK-1-32 were selected for further studies. Neuronal and microglial viabilities were assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay using neuroblastoma (SHSY-5Y) and human microglial (HMC-3) cell lines, respectively. Lipopolysaccharide (LPS)-induced inflammation in HMC-3 cells was used for neurotoxicity assay. Other assays include nitric oxide (NO) by Griess reagent, cofilin inhibition by F-actin depolymerization, migration by scratch wound assay, tumor necrosis factor (TNF-α) by enzyme-linked immunosorbent assay (ELISA), protease-activated receptor-1 (PAR-1) by immunocytochemistry and Western blotting (WB), and protein expression levels of several proteins by WB. SK-1-32 increased neuronal/microglial survival, reduced NO, and prevented neurotoxicity. However, SZ-3 showed no effect on neuronal/microglial survival but prevented microglia from LPS-induced inflammation by decreasing NO and preventing neurotoxicity. Therefore, we selected SZ-3 for further molecular studies, as it showed potent anti-inflammatory activities. SZ-3 decreased cofilin severing activity, and its treatment of LPS-activated HMC-3 cells attenuated microglial activation and suppressed migration and proliferation. HMC-3 cells subjected to thrombin, as an in vitro model for hemorrhagic stroke, and treated with SZ-3 after 3 h showed significantly decreased NO and TNF-α, significantly increased protein expression of phosphocofilin, and decreased PAR-1. In addition, SZ-3-treated SHSY-5Y showed a significant increase in cell viability by significantly reducing nuclear factor-κ B (NF-κB), caspase-3, and high-temperature requirement (HtrA2). Together, our results support the novel idea of targeting cofilin to counter neuroinflammation during secondary injury following ICH.

Type-I diabetes aggravates post-hemorrhagic stroke cognitive impairment by augmenting oxidative stress and neuroinflammation in mice.

Diabetes Mellitus (DM) is a major comorbid condition that increases susceptibility to stroke. Intracerebral hemorrhage (ICH), a devastating type of stroke, accounts for only 13% of the total stroke cases but is associated with higher mortality. Multimorbid models of DM and ischemic stroke have been widely studied; however, fewer pieces of evidence are available on the impact of DM on the outcomes of ICH injury. In this study, we investigated the effect of DM on ICH-induced injury and cognitive impairments. Streptozotocin (STZ) induced type-I DM (T1DM) animal model was used, and experimental ICH was induced by intrastriatal injection of collagenase. Our results demonstrated that DM is associated with a significant increase in hematoma volume and deficits in post-stroke locomotor, sensorimotor, and cognitive behavior in mice. The levels of neuroinflammation, oxidative/nitrosative stress, and glial cell activation were also increased in the diabetic mice following ICH injury. This study provides a better understanding of the influence of DM comorbidity on hemorrhagic stroke outcomes and uncovers the important pathological mechanisms underlying DM-induced exacerbation of ICH injury.

Potential Benefits of N-Acetylcysteine in Preventing Pregabalin-Induced Seeking-Like Behavior.

Substance-use disorder is globally prevalent and responsible for numerous social and medical problems. Pregabalin (Lyrica), typically used to treat diabetic neuropathy, has recently emerged as a drug of abuse. Drug abuse is associated with several neuronal changes, including the downregulation of glutamate transporters such as glutamate transporter 1 and cystine/glutamate antiporter. We investigated the effects of N-acetylcysteine, a glutamate transporter 1 and xCT upregulator, on pregabalin addiction using a conditioned place preference paradigm. Pregabalin (60 mg/kg) was found to induce conditioned place preference when compared to a vehicle. A 100 mg/kg dose of N-acetylcysteine was found to block pregabalin-seeking behaviors. These results support previous findings showing that glutamate transporters play an important role in pregabalin-induced seeking behaviors. N-acetylcysteine may represent a beneficial agent in preventing the abuse potential of pregabalin.

Generating homogenous cortical preplate and deep-layer neurons using a combination of 2D and 3D differentiation cultures.

Embryonic stem cells (ESCs) can be used to derive different neural subtypes. Current differentiation protocols generate heterogeneous neural subtypes rather than a specific neuronal population. Here, we present a protocol to derive separate two-deep layer cortical neurons from mouse ESCs (mESCs). mESCs were differentiated into mature Tbr1 or Ctip2-positive neurons using a monolayer-based culture for neural induction and neurosphere-based culture for neural proliferation and expansion. The differentiation protocol relies on SMAD inhibition for neural induction and the use of FGF2 and EGF for proliferation and it is relatively short as mature neurons are generated between differentiation days 12-16. Compared with the monolayer-based differentiation method, mESCs can be directed to generate specific deep-layer cortical neurons rather than heterogeneous cortical neurons that are generated using the monolayer differentiation culture. The early analysis of progenitors using flow cytometry, immunocytochemistry, and qRT-PCR showed high neuralization efficiency. The immunocytochemistry and flow cytometry analyses on differentiation days 12 and 16 showed cultures enriched in Tbr1- and Ctip2-positive neurons, respectively. Conversely, the monolayer differentiation culture derived a mixture of Tbr1 and Ctip2 mature neurons. Our findings suggested that implementing a neurosphere-based culture enabled directing neural progenitors to adopt a specific cortical identity. The generated progenitors and neurons can be used for neural-development investigation, drug testing, disease modelling, and examining novel cellular replacement therapy strategies.

Pregabalin: Potential for Addiction and a Possible Glutamatergic Mechanism.

Drug addiction remains a prevalent and fatal disease worldwide that carries significant social and economic impacts. Recent reports suggest illicit pregabalin (Lyrica) use may be increasing among youth, however the addictive potential of pregabalin has not been well established. Drug seeking behavior and chronic drug use are associated with deficits in glutamate clearance and activation of postsynaptic glutamatergic receptors. In the current study, we investigated the abuse potential of pregabalin using conditioned place preference (CPP) paradigm. Different doses of pregabalin (30, 60, 90, and 120 mg/kg) were used to assess the seeking behavior in mice. Glutamate homeostasis is maintained by glutamate transporter type-1 (GLT-1), which plays a vital role in clearing the released glutamate from synapses and drug seeking behavior. Therefore, we investigated the role of glutamate in pregabalin-seeking behavior with ceftriaxone (CEF), a potent GLT-1 upregulator. Mice treated with pregabalin 60 and 90 mg/kg doses demonstrated drug seeking-like behavior, which was significantly blocked by CEF pretreatment. These results suggest that pregabalin-induced CPP was successfully modulated by CEF which could serve as a lead compound for developing treatment for pregabalin abuse.

Tribulusterine Containing Tribulus terrestris Extract Exhibited Neuroprotection Through Attenuating Stress Kinases Mediated Inflammatory Mechanism: In Vitro and In Vivo Studies.

The present study has been aimed to explore the different secondary messengers of the inflammatory pathway NF-κB, kinases (JNK, P38MAPK, GSK3ß/ßcatenin), apoptosis pathway (Caspase-3 and AIF), and neuronal survival pathway (BDNF) in order to understand the neuroprotective mechanism of aqueous extract of Tribulus terrestris (AQTT). In primary cortical neurons, the ischemic condition was induced through oxygen-glucose deprivation (OGD). Anti-inflammatory activity of AQTT was evaluated in formalin induced inflammation model and carrageenan-induced paw edema test. The bilateral common carotid artery occlusion model was employed for whole animal studies. Treatment of AQTT (100 mg/kg) significantly reduced the inflammation induced by formalin and carrageenan. The neuroprotective mechanism of AQTT (50 and 100 mg/kg) was assessed by pre- and post-administration. The results indicate down regulation of kinases and NFkB, suggesting possible anti-inflammatory activity of AQTT. Additionally, AQTT down regulated both caspase dependent and independent apoptotic pathways suggesting its possible anti-apoptotic activity. The treatment of AQTT also reduced GSK3ß levels and increased p-Ser9 GSK3ß levels; stabilizing the unphosphorylated form of ß-catenin and its translocation into the nucleus suggesting role of AQTT in neuronal survival and GSK3ß mediated anti-inflammatory property. In comparison to pretreatment, post treatment of AQTT had lesser effects indicating tribulusterine standardized AQTT may have prophylactic effect. This study can be concluded with the thesis that AQTT has neuroprotective effect through alternating neuroinflammation, apoptosis, and promoting neuron survival. Being that it produced better effect with pretreatment, exploring this with thrombolytic drugs will be beneficial. For the first time AQTT has been reported for this indication.

A Bioactive Resveratrol Trimer from the Stem Bark of the Sri Lankan Endemic Plant Vateria copallifera.

A new resveratrol trimer, vateriferol (1), having four cis-oriented methine protons and constituting four contiguous stereocenters, was isolated from the bark extract of Vateria copallifera by bioassay-guided fractionation using a combination of normal, reversed phase, and size exclusion column chromatography. The structure was established based on its spectroscopic data. Vateriferol (1) was evaluated in vitro for its antioxidant capacity, enzyme inhibitory activity, growth inhibitory activity on a number of cancer cell lines, neuroprotective activity, and anti-inflammatory activity. Vateriferol (1) exhibited AChE inhibitory activity (IC50 8.4 ± 0.2 µM), ORAC activity (2079 ± 0.20 TE/g), and neuroprotective activity at 1.5 µM using PC12 cells deprived of oxygen and glucose and lowered NO levels in lipopolysaccharide-stimulated SIM-A9 microglial cells at 14.7 and 73.6 µM. Vateriferol (1) exhibited weak cytotoxic potency (<50% growth inhibition) against the tested cell lines at 147.2 µM.

Cofilin Knockdown Attenuates Hemorrhagic Brain Injury-induced Oxidative Stress and Microglial Activation in Mice.

Intracerebral hemorrhage (ICH) resulting from the rupture of the blood vessels in the brain is associated with significantly higher mortality and morbidity. Clinical studies focused on alleviating the primary injury, hematoma formation and expansion, were largely ineffective, suggesting that secondary injury-induced inflammation and the formation of reactive species also contribute to the overall injury process. In this study, we explored the effects of cofilin knockdown in a mouse model of ICH. Animals given stereotaxic injections of cofilin siRNA, 72-h prior to induction of ICH by collagenase injection within the area of siRNA administration showed significantly decreased cofilin expression levels and lower hemorrhage volume and edema, and the animals performed significantly better in neurobehavioral tasks i.e., rotarod, grip strength and neurologic deficit scores. Cofilin siRNA knocked-down mice had reduced ICH-induced DNA fragmentation, blood-brain barrier disruption and microglial activation, with a concomitant increase in astrocyte activation. Increased expression of pro-survival proteins and decreased markers of oxidative stress were also observed in cofilin siRNA-treated mice possibly due to the reduced levels of cofilin. Our results suggest that cofilin plays a major role in ICH-induced secondary injury, and could become a potential therapeutic target.

Furoxans (Oxadiazole-4 N-oxides) with Attenuated Reactivity are Neuroprotective, Cross the Blood Brain Barrier, and Improve Passive Avoidance Memory.

Nitric oxide (NO) mimetics and other agents capable of enhancing NO/cGMP signaling have demonstrated efficacy as potential therapies for Alzheimer's disease. A group of thiol-dependent NO mimetics known as furoxans may be designed to exhibit attenuated reactivity to provide slow onset NO effects. The present study describes the design, synthesis, and evaluation of a furoxan library resulting in the identification of a prototype furoxan, 5a, which was profiled for use in the central nervous system. Furoxan 5a demonstrated negligible reactivity toward generic cellular thiols under physiological conditions. Nonetheless, cGMP-dependent neuroprotection was observed, and 5a (20 mg/kg) reversed cholinergic memory deficits in a mouse model of passive avoidance fear memory. Importantly, 5a can be prepared as a pharmaceutically acceptable salt and is observed in the brain 12 h after oral administration, suggesting potential for daily dosing and excellent metabolic stability. Continued investigation into furoxans as attenuated NO mimetics for the CNS is warranted.

Cofilin Mediates LPS-Induced Microglial Cell Activation and Associated Neurotoxicity Through Activation of NF-κB and JAK-STAT Pathway.

Microglial cells are activated in response to different types of injuries or stress in the CNS. Such activation is necessary to get rid of the injurious agents and restore tissue homeostasis. However, excessive activation of microglial cells is harmful and contributes to secondary injury. Pertinently, microglial cell activity was targeted in many preclinical and clinical studies but such strategy failed in clinical trials. The main reason behind the failed attempts is the complexity of the injury mechanisms which needs either a combination therapy or targeting a process that is involved in multiple pathways. Cofilin is a cytoskeleton-associated protein involved in actin dynamics. In our previous study, we demonstrated the role of cofilin in mediating neuronal apoptosis during OGD conditions. Previous studies on microglia have shown the involvement of cofilin in ROS formation and phagocytosis. However, additional studies are needed to delineate the role of cofilin in microglial cell activation. Therefore, in the current study, we investigated the role of cofilin in LPS-induced microglial cell activation using cofilin siRNA knockdown paradigms. The viability of differentiated PC12 cells was used as a measure of the neurotoxic potential of conditioned medium derived from cofilin siRNA-transfected and LPS-activated microglial cells. Cofilin knockdown significantly inhibited LPS-induced microglial cell activation through NF-κB and JAK-STAT pathways. The release of proinflammatory mediators (NO, TNF-α, iNOS, and COX2) as well as microglial proliferation and migration rates were significantly reduced by cofilin knockdown. Furthermore, differentiated PC12 cells were protected from the neurotoxicity induced by conditioned medium derived from cofilin-transfected and LPS-activated microglial cells. In conclusion, we demonstrated that cofilin is involved in the cascade of microglial cell activation and further validates our previous study on cofilin's role in mediating neuronal apoptosis. Together, our results suggest that cofilin could present a common target in neurons and microglial cells and might prove to be a promising therapy for different brain injury mechanisms including stroke.

Cofilin signaling in hemin-induced microglial activation and inflammation.

Intracerebral hemorrhage (ICH) is the most severe form of stroke and is further exacerbated by the secondary injury involving inflammatory response due to the activation of microglia. This secondary injury is partly due to the toxic effects of hemin, an endogenous breakdown product of hemoglobin. Cofilin, an actin depolymerizing factor, controls actin dynamics and has been previously shown to be involved in mediating neuronal cell death in ischemic conditions and during bacterial lipopolysaccharide induced microglial activation. There are limited studies regarding the deleterious effects of extremely high concentrations of hemin released during ICH and its effects on microglia and subsequent cofilin response. Therefore, investigations were conducted to study the effects of hemin on microglial activation induced inflammation and the critical role of cofilin in mediating the response. We observed that hemin treated microglia had a concentration dependent increase in cofilin expression and NO production. There were increased levels of iNOS, TNF-α, HO1, Nrf2, Wfs-1, XBP-1 and spliced XBP-1 observed in response to hemin treatment and the signaling was found to be partly mediated by cofilin. Acute hemin treatment did not evoke Ca2+ signaling and long-term treatment of hemin also resulted in the failure of microglial response to acetylcholine-evoked Ca2+ signaling. Knockdown of cofilin by siRNA also reduced acetylcholine-evoked Ca2+ signaling. These studies demonstrate that cofilin signaling is important in hemin-induced inflammation, oxidative stress, ER stress, microglial migration, and the ability to evoke Ca2+ signaling. Therefore, cofilin inhibition could be a potential therapy in brain injuries triggered by hemin toxicity in conditions like ICH.

Tanshinone IIA Inhibits VEGF Secretion and HIF-1α Expression in Cultured Human Retinal Pigment Epithelial Cells under Hypoxia.

PURPOSE: The current work intends to study the activity of tanshinone IIA on secretion of vascular endothelial growth factor (VEGF) and expression of hypoxia inducible factor 1α (HIF-1α) in human retinal pigment epithelial cells (ARPE-19 cells) under hypoxic condition. METHODS: The cytotoxicity of tanshinone IIA was tested in ARPE-19 cells by MTT assay. ARPE-19 cells were incubated with different concentrations of cobalt chloride (100, 150, and 200 µM) for 12 h, and levels of expressed HIF-1α and secreted VEGF were quantified through Western blot and ELISA, respectively. Further, ARPE-19 cells were pretreated for 1 h with different concentrations of tanshinone IIA (5, 10, 15, and 18 µM). After 1 h, the cells were subjected to hypoxic condition using 150 µM cobalt chloride for 12 h in the presence and absence of tanshinone IIA. The cells were then harvested, and the secreted VEGF and expressed HIF-1α was studied. RESULTS: Tanshinone IIA at concentrations of 5, 10, 15, and 18 µM did not show cytotoxicity in ARPE-19 cells. Chemical hypoxia induced by cobalt chloride caused a significant increase in VEGF level in a dose-dependent manner, and HIF-1α expression peaked at 150 µM. Based on the data, cobalt chloride concentration was maintained at 150 µM for further studies. Tanshinone IIA decreased the level of HIF-1α and VEGF secretion in a dose-dependent manner under hypoxic condition. CONCLUSION: Tanshinone IIA could be explored as a new potential candidate for treating wet AMD.

The Interplay between Cofilin and Phospho-Cofilin: Its Role in Maintaining Blood Brain Barrier Integrity.

The blood brain barrier (BBB) is a continuous, non-fenestrated vessel system that tightly regulates the movement of molecules, ions, and cells between the blood and the central nervous system. Endothelial cells are the major constituents of the BBB and these cells are linked to each other through intercellular contact points composed of tight junctions, adherent junctions and gap junctions. These three types of junctions are connected to the intracellular actin cytoskeleton via various adaptor proteins. Thus, the actin cytoskeleton plays a crucial role in regulating the stability of endothelial cell contacts and vascular permeability. Shear stress, growth factors, and Wnt/ß-catenin pathway modulators contribute to maintaining endothelial cell integrity by controlling actin dynamics under homeostatic conditions. Interestingly, the downstream signaling of the aforementioned factors converges at Rac1, which mediates cortical actin stabilization, stress fiber destabilization and junctional complex stabilization by controlling subcellular cofilin dynamics. However, Rac1 is not the only modulator of cofilin activity; many other agents activated during inflammatory, ischemic, and excitotoxic conditions can disturb homeostatic cofilin dynamics and induce BBB disruption. Therefore, in this review, we discuss organization of the actin cytoskeleton in BBB endothelial cells and how interactions between the actin cytoskeleton and junctional complexes are maintained during homeostatic conditions. Furthermore, we discuss how an imbalance in subcellular cofilin dynamics can contribute to BBB disruption and highlight Rac1 as a potential target that can be exploited to preserve BBB stability.

Natural Polyphenols in Cancer Chemoresistance.

Resistance to chemotherapy remains a major impediment to the management of most types of cancer. Both intrinsic and acquired drug resistance are mediated by several cellular and molecular mechanisms, including alternative growth-signaling pathways unaffected by specific therapies, alterations in the tumor microenvironment (e.g., hypoxia and angiogenesis), and active transport of drugs out of the cell. Epidemiological studies have validated an inverse correlation between the consumption of dietary polyphenols and the risk of cancer, which has been attributed to polyphenol antioxidant capacity and their potential to inhibit activation of procarcinogens, cancer cell proliferation, metastasis, and angiogenesis, and inhibition or downregulation of active drug efflux transporters. Moreover, polyphenols can induce apoptosis in cancer cells and modulate immune responses and inflammatory cascades. Augmentation of the efficacy of chemotherapy and prevention of multidrug resistance are other important effects of dietary polyphenols that deserve further research, especially after the discovery of tight "crosstalk" between aberrant growth signaling and metabolic dysfunction in cancer cells. In this review, we cover what is currently known about the role of natural polyphenolic compounds in overcoming cancer drug resistance mediated by diverse primary and secondary resistance mechanisms.

Natural polyphenols: Influence on membrane transporters.

Accumulated evidence has focused on the use of natural polyphenolic compounds as nutraceuticals since they showed a wide range of bioactivities and exhibited protection against variety of age-related disorders. Polyphenols have variable potencies to interact, and hence alter the activities of various transporter proteins, many of them classified as anion transporting polypeptide-binding cassette transporters like multidrug resistance protein and p-glycoprotein. Some of the efflux transporters are, generally, linked with anticancer and antiviral drug resistance; in this context, polyphenols may be beneficial in modulating drug resistance by increasing the efficacy of anticancer and antiviral drugs. In addition, these effects were implicated to explain the influence of dietary polyphenols on drug efficacy as result of food-drug interactions. However, limited data are available about the influence of these components on uptake transporters. Therefore, the objective of this article is to review the potential efficacies of polyphenols in modulating the functional integrity of uptake transporter proteins, including those terminated the effect of neurotransmitters, and their possible influence in neuropharmacology.

Cofilin as a Promising Therapeutic Target for Ischemic and Hemorrhagic Stroke.

Neurovascular unit (NVU) is considered as a conceptual framework for investigating the mechanisms as well as developing therapeutic targets for ischemic and hemorrhagic stroke. From a molecular perspective, oxidative stress, excitotoxicity, inflammation, and disruption of the blood brain barrier are broad pathophysiological frameworks on the basis on which potential therapeutic candidates for ischemic and hemorrhagic stroke could be discussed. Cofilin is a potent actin-binding protein that severs and depolymerizes actin filaments in order to generate the dynamics of the actin cytoskeleton. Although studies of the molecular mechanisms of cofilin-induced reorganization of the actin cytoskeleton have been ongoing for decades, the multicellular functions of cofilin and its regulation in different molecular pathways are expanding beyond its primary role in actin cytoskeleton. This review focuses on the role of cofilin in oxidative stress, excitotoxicity, inflammation, and disruption of the blood brain barrier in the context of NVU as well as how and why cofilin could be studied further as a potential target for ischemic and hemorrhagic stroke.