Search

High-mobility group box 1 protein (HMGB1) is a damage-associated molecular pattern (DAMP) involved in neutrophil extracellular trap (NET) formation and thrombosis. NETs are regularly found in cerebral thromboemboli. We here analyzed associated HMGB1 expression in human thromboemboli retrieved via mechanical thrombectomy from 37 stroke patients with large vessel occlusion. HMGB1 was detected in all thromboemboli, accounting for 1.7% (IQR 0.6-6.2%) of the total thromboemboli area and was found to be colocalized with neutrophils and NETs and in spatial proximity to platelets. Correlation analysis revealed that the detection of HMGB1 was strongly related to the number of neutrophils (r = 0.58, p = 0.0002) and platelets (r = 0.51, p = 0.001). Our results demonstrate that HMGB1 is a substantial constituent of thromboemboli causing large vessel occlusion stroke.

Objectives: Thrombi responsible for large vessel occlusion (LVO) in the setting of acute ischemic stroke (AIS) are characterized by a low recanalization rate after IV thrombolysis. To test whether AIS thrombi have inherent common features that limit their susceptibility to thrombolysis, we analyzed the composition and ultrastructural organization of AIS thrombi causing LVO., Methods: A total of 199 endovascular thrombectomy-retrieved thrombi were analyzed by immunohistology and scanning electron microscopy (SEM) and subjected to ex vivo thrombolysis assay. The relationship between thrombus organization and thrombolysis resistance was further investigated in vitro using thrombus produced by recalcification of citrated whole blood., Results: SEM and immunohistology analyses revealed that, although AIS thrombus composition and organization was highly heterogeneous, AIS thrombi shared a common remarkable structural feature in the form of an outer shell made of densely compacted thrombus components including fibrin, von Willebrand factor, and aggregated platelets. In vitro thrombosis experiments using human blood indicated that platelets were essential to the formation of the thrombus outer shell. Finally, in both AIS and in vitro thrombi , the thrombus outer shell showed a decreased susceptibility to tissue plasminogen activator-mediated thrombolysis as compared to the thrombus inner core., Interpretation: Irrespective of their etiology and despite their heterogeneity, intracranial thrombi causing LVO have a core shell structure that influences their susceptibility to thrombolysis., (© 2019 American Academy of Neurology.)

Background and Purpose- A major process contributing to cell death in the ischemic brain is inflammation. Inflammasomes are multimolecular protein complexes that drive inflammation through activation of proinflammatory cytokines, such as IL (interleukin)-1β. Preclinical evidence suggests that IL-1β contributes to a worsening of ischemic brain injury. Methods- Using a mouse middle cerebral artery thrombosis model, we examined the inflammatory response after stroke and the contribution of the NLRP3 (NACHT, LRR and PYD domains-containing protein 3) inflammasome to ischemic injury. Results- There was a marked inflammatory response after stroke characterized by increased expression of proinflammatory cytokines and NLRP3 and by recruitment of leukocytes to the injured tissue. Targeting NLRP3 with the inhibitor MCC950, or using mice in which NLRP3 was knocked out, had no effect on the extent of injury caused by stroke. Conclusions- These data suggest that the NLRP3 pathway does not contribute to the inflammation exacerbating ischemic brain damage, contradicting several recent reports to the contrary.

Present investigation evaluates the protective effect of Melilotus officinalis (MO) extract on the brain tissues in acute cerebral ischemia. Acute cerebral ischemia was induced by occlusion of carotid artery and rats with cerebral ischemia were treated with MO (100, 250 & 500mg/kg) for the duration of three days. Cerebral ischemia was confirmed by estimating infract volume and neurological deficit score. Moreover biochemical parameters in plasma such as 6-keto-PGF1α and TXB2 and concentration of cytokine, oxidative stress, apoptosis ratio and protein expressions of Bcl2 & Bax were estimated in the brain tissues. It was observed that treatment with MO significantly (p<0.01) decreases the infract volume and neurological deficit score than negative control group. There was significant decrease (p<0.01) in the oxidative stress and cytokine in the brain tissues and increase in the plasma concentration of 6-keto-PGF1α in MO treated group of rats compared to negative control group. Plasma concentration of TXB 2 was significantly enhanced in MO treated group compared to negative control group of rats. It was also found that treatment with MO ameliorates the apoptosis induced by cerebral ischemia. Present study concludes that MO ameliorates apoptosis of brain tissues in cerebral ischemic rats by decreasing cerebral thrombosis, oxidative stress and inflammatory mediators., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Background: Understanding the underlying mechanism of thrombus formation and its components is critical for effective prevention and treatment of ischemic stroke. The generation of thrombotic clots requires conversion of soluble fibrinogen to an insoluble fibrin network. Quantitative features of intracranial clots causing acute ischemic stroke can be studied on non-contrast enhanced CT (NECT). Here, we evaluated on-admission fibrinogen and clot burden in relation to stroke severity, final infarct volume and in-hospital mortality., Methods: We included 132 consecutive patients with ischemic stroke and presence of hyperdense artery sign admitted within 6 h from symptom onset. Radiological parameters including clot area (corresponding to clot burden) and final infarct volume were manually determined on NECT. National Institute of Health Stroke Scale (NIHSS) was used to quantify disease severity and short-term outcome., Results: Median patient age was 77, 58 % were women, and 63 % had an occlusion of the proximal middle cerebral artery segment. Thrombolysis was performed in 60 % and thrombectomy in 44 %. We identified several independent associations. Higher fibrinogen levels on admission were associated with smaller clot burden (p = 0.033) and lower NIHSS on admission (p = 0.022). Patients with lower fibrinogen had a higher clot burden (p = 0.028) and greater final infarct volume (p = 0.003). Higher fibrinogen was associated with a lower risk of in-hospital death or NIHSS score >15 if discharged alive (p = 0.028)., Conclusions: Our study suggests that intracranial clot burden in acute ischemic stroke is associated with fibrinogen consumption, and shows a complex relationship with disease severity, infarct size and in-hospital survival.

Ethnopharmacological Relevance: Polygonum multiflorum Thunb. has been used widely in East Asia in treatment of diseases associated with aging. Emodin, an active component from Polygonum multiflorum Thunb., provides benefits for brain disturbances induced by severe cerebral injury., Aim of the Study: We investigated the neuroprotective effect of emodin from Polygonum multiflorum Thunb. against glutamate-induced oxidative toxicity and cerebral ischemia., Materials and Methods: For examination of neuroprotective effects of emodin, cell viability, cytotoxicity, flow cytometry, and Western blot were performed in HT22 cells and infarct volume, behavioral tests and Western blot in a mouse model of photothrombotic ischemic stroke., Results: Pretreatment with emodin resulted in significantly reduced glutamate-induced apoptotic cell death in HT22 cells. However, blocking of phosphatidylinositol-3 kinase (PI3K) activity with LY294002 resulted in significantly inhibited cell survival by emodin. Exposure of glutamate-treated cells to emodin induced an increase in the level of Bcl-2 expression, whereas the expression of Bax and active caspase-3 proteins was significantly reduced. In addition, treatment with emodin resulted in increased phosphorylation of Akt and cAMP response element binding protein (CREB), and expression of mature brain-derived neurotrophic factor (BDNF). This expression by emodin was also significantly inhibited by blocking of PI3K activity. In a photothrombotic ischemic stroke model, treatment with emodin resulted in significantly reduced infarct volume and improved motor function. We confirmed the critical role of the expression levels of Bcl-2/Bax, active caspase-3, phosphorylated (p)Akt, p-CREB, and mature BDNF for potent neuroprotective effects of emodin in cerebral ischemia., Conclusions: These results suggest that emodin may afford a significant neuroprotective effect against glutamate-induced apoptosis through activation of the PI3K/Akt signaling pathway, and subsequently enhance behavioral function in cerebral ischemia., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Photodynamic impact on animal cerebral cortex using water-soluble Bengal Rose as a photosensitizer, which does not cross the blood-brain barrier and remains in blood vessels, induces platelet aggregation, vessel occlusion, and brain tissue infarction. This reproduces ischemic stroke. Irreversible cell damage within the infarction core propagates to adjacent tissue and forms a transition zone - the penumbra. Tissue necrosis in the infarction core is too fast (minutes) to be prevented, but much slower penumbral injury (hours) can be limited. We studied the changes in morphology and protein expression profile in penumbra 1 h after local photothrombotic infarction induced by laser irradiation of the cerebral cortex after Bengal Rose administration. Morphological study using standard hematoxylin/eosin staining showed a 3-mm infarct core surrounded by 1.5-2.0 mm penumbra. Morphological changes in the penumbra were lesser and decreased towards its periphery. Antibody microarrays against 224 neuronal and signaling proteins were used for proteomic study. The observed upregulation of penumbra proteins involved in maintaining neurite integrity and guidance (NAV3, MAP1, CRMP2, PMP22); intercellular interactions (N-cadherin); synaptic transmission (glutamate decarboxylase, tryptophan hydroxylase, Munc-18-1, Munc-18-3, and synphilin-1); mitochondria quality control and mitophagy (PINK1 and Parkin); ubiquitin-mediated proteolysis and tissue clearance (UCHL1, PINK1, Parkin, synphilin-1); and signaling proteins (PKBα and ERK5) could be associated with tissue recovery. Downregulation of PKC, PKCβ1/2, and TDP-43 could also reduce tissue injury. These changes in expression of some neuronal proteins were directed mainly to protection and tissue recovery in the penumbra. Some upregulated proteins might serve as markers of protection processes in a penumbra.

Focal cerebral ischemia is among the main causes of death and disability worldwide. The ischemic core often progresses, invading the peri-ischemic brain; however, assessing the propensity of the peri-ischemic brain to undergo secondary damage, understanding the underlying mechanisms, and adjusting treatment accordingly remain clinically unmet challenges. A significant hallmark of the peri-ischemic brain is dysfunction of the blood-brain barrier (BBB), yet the role of disturbed vascular permeability in stroke progression is unclear. Here we describe a longitudinal in vivo fluorescence imaging approach for the evaluation of cortical perfusion, BBB dysfunction, free radical formation and cellular injury using the photothrombosis vascular occlusion model in male Sprague Dawley rats. Blood-brain barrier dysfunction propagated within the peri-ischemic brain in the first hours after photothrombosis and was associated with free radical formation and cellular injury. Inhibiting free radical signaling significantly reduced progressive cellular damage after photothrombosis, with no significant effect on blood flow and BBB permeability. Our approach allows a dynamic follow-up of cellular events and their response to therapeutics in the acutely injured cerebral cortex.

Stroke is one of the leading causes of death worldwide. Formation of a fibrin clot is controlled by a group of tightly regulated plasma proteases and cofactors and a change in the fibrin fiber formation causes an alteration in clot morphology. This plays an important role during thrombotic events. In the current study we investigated the ultrastructure of fibrin networks from fifteen ischemic stroke patients by using scanning electron microscopy. Clot morphology was investigated with and without the addition of human thrombin to the platelet rich plasma. Previously it was shown that, when studying the ultrastructure of fibrin networks, the addition of thrombin is necessary to form an expansive, fully coagulated layer of fibers. Results from the addition of thrombin to the plasma showed thick, matted fibrin fibers and a net covering some of the major fibers in stroke patients. Typical control morphology with major thick fibers and minor thin fibers could be seen in some areas in the stroke patients. In stroke patients, without the addition of thrombin, a matted fibrin network still formed, indicating that the factors responsible for the abnormal fibrin morphology are present in the circulating plasma and is the cause of the observed matted, layered morphology. This is not present in healthy individuals. From the results obtained we suggest that this changed morphology might be useful in a screening regime to identify the possibility of a stroke or even to follow the progress of stroke patients after treatment.

Perifocal ischemia zone ultrastructural changes caused by unilateral photoinduced thrombosis of the rat prefrontal brain and reciprocal changes in an opposite hemisphere of a brain in 1 and 24 hours after an ischemia are investigated. Reactive changes of microcirculation, astrocytes, neurons in the perifocal zone of the ischemia, and also in the contralateral site of the intact hemisphere as increase in hematoencephalic barrier permeability, numbers of degraded neurons, astrocyte transport injury and glucose metabolic imbalance are revealed. The degree of reactive changes of neurons and glia in the contralateral hemisphere is less expressed, than in a right hemisphere and has other temporal dynamics.

The aim of this study was to investigate the influence of delayed Rho-kinase inhibition with fasudil on second ischemic injury in a rat cerebral thrombosis model. Cerebral ischemia was induced in rats by injecting 150 mug of sodium laurate into the left internal carotid artery on day 1. In the ischemic group, the regional cerebral blood flow (rCBF) was significantly decreased 6.5 h after the injection. Fasudil (3 mg/kg/30 min i.v. infusion) significantly increased rCBF. The viscosity of whole blood was significantly increased 48 h after the injection of sodium laurate. Fasudil (10 mg/kg, i.p.) significantly decreased blood viscosity. To clarify the therapeutic time window of fasudil, rats received their first i.p. administration of fasudil (10 mg/kg) 6 h after an injection of sodium laurate. Administration of fasudil twice daily was continued until day 4. Fasudil prevented the accumulation of neutrophils within the brain as seen from measurements taken on day 3, and improved neuronal functions and reduced the infarction area as seen on day 5. Fasudil and hydroxyfasudil, an active metabolite of fasudil, concentration-dependently inhibited phosphorylation of myosin binding subunit of myosin phosphatase in neutrophils. The present results indicate that inhibition of Rho-kinase activation with fasudil is effective for the treatment of ischemic brain damage with a wide therapeutic time window by improving hemodynamic function and preventing the inflammatory responses. These results suggest that fasudil will be a novel and efficacious approach for the treatment of acute ischemic stroke.

Photothrombotic model of ischemia (PT) is based on free radical-mediated endothelial dysfunction followed by thrombosis. Free radicals are also involved in hypoxic preconditioning. We tested the sensitivity of PT to preconditioning with hypobaric hypoxia and to pretreatment with melatonin. In adult Wistar rats, after intravenous application of Rose Bengal, a stereo-tactically defined spot on the denuded skull was irradiated by a laser for 9 min. The first experimental group underwent hypobaric hypoxia three days before irradiation. In the second experimental group, melatonin was applied intraperitoneally one hour before irradiation. Three days after irradiation, animals were sacrificed, the brains perfused, and stained with TTC. Ischemic lesions were divided into grades (I, II, III). In the control group (where no manipulation preceded photothrombosis), most animals displayed deep damage involving the striatum (grade III). The group pre-exposed to hypoxia showed similar results. Only 28.57 % of the melatonin pretreated animals exhibited grade III lesions, and in 57.14 % no signs of lesions were detected. Pre-exposure to hypoxia was not protective in our model. Pretreatment with melatonin lead to a significant reduction of the number of large ischemic lesions. This result is probably caused by protection of endothelial cells by melatonin.

Several types of CNS injury and various diseases are associated with the development of a glial scar. Astrocytes are major components of the glial scar. They are interconnected by gap junctions, with connexin43 (Cx43) being the most prominent channel protein. We applied a model of focal cerebral ischemia to study the spatio-temporal expression of glial fibrillary acidic protein, as well as of Cx43 mRNA and protein in gliotic tissue up to 60 days after injury. Reactive astrocytes enveloping the lesion up-regulated their Cx43 mRNA and protein. A band of reactive astrocytes filling in the lesion exhibited elevated Cx43 and showed a high degree of proliferation. Because of these findings, we hypothesize a role for Cx43 in glial scar formation, specifically in the proliferation of astrocytes.

E-selectin, which is a member of the selectin superfamily of adhesion molecules, contributes to the leukocyte-endothelial cell attachments and is involved in the pathogenesis of thrombovascular diseases as a consequence. We investigated the A561C mutation in the E-selectin gene in 235 Japanese patients with ischemic cerebrovascular disease (CVD) and 301 age- and sex-matched healthy controls. Excluding the subjects with diabetes mellitus and hypercholesterolemia, the AC genotype frequencies of patients with ischemic CVD were higher than those of controls: 12.7% vs. 5.8% (P=0.04). Our results show that E-selectin gene polymorphisms represent an increased risk for ischemic CVD in the Japanese population without diabetes mellitus and hypercholesterolemia.

Focal cerebral lesions in rat brain induced by photothrombosis lead to an impaired inhibitory neurotransmission. A reduced gamma-aminobutyric acid (GABA)-mediated inhibition has been revealed by electrophysiological recordings associated with a diminished immunostaining of GABA handling proteins. Changes were found in ipsi- as well as in contralateral brain areas. Inhibition is mediated by interneurons using GABA as neurotransmitter. These cells use GAD (glutamate decarboxylase) to synthesize GABA. To analyze the vulnerability of GABAergic neurons in rats with a lesioned hindlimb area, cells expressing GAD65/67 mRNA were labeled using in situ hybridization. Positive somata were counted 7 and 30 days after focal ischemia in different cortical (hindlimb cortex, frontal cortex, primary and secondary somatosensory cortex) and hippocampal subsectors (pyramidal cell layer, stratum oriens and stratum radiatum/lacunosum-moleculare). The lesioned hemispheres were compared with the intact brain sides and with control brains. GABAergic interneurons survived the injury for up to 30 days in all investigated brain regions. Therefore it is unlikely that a loss of GABAergic neurons contributes to the reduced inhibition.

The impact of species-specific factors on postischemic brain inflammation is largely unknown. In this study, we used quantitative real-time polymerase chain reaction in a highly standardized model of focal cortical brain ischemia for the comparison of cytokine and inducible nitric oxide synthase (iNOS) gene expression in rats and mice. In rats, we found rapid and strong induction of tumor necrosis factor-alpha (TNF-alpha) mRNA reaching its peak at 4 h after ischemia, followed by a slightly delayed peak of interleukin-1beta (IL-1beta) and iNOS mRNA at 16 h. Inflammatory gene induction in mice was overall weaker and considerably more protracted. Both TNF-alpha and IL-1beta transcripts reached their peak around 24 h. In addition, iNOS mRNA exhibited a rather variable, delayed increase between days 3 and 14. Accordingly, immunocytochemistry revealed strong iNOS immunoreactivity in the infarct borderzone at days 1 and 3 in rats whereas only a few iNOS-positive cells were detectable in mice. Taken together, our study demonstrates considerable species differences in inflammatory gene induction after focal brain ischemia.

The role of endogenous tissue-type plasminogen activator (tPA) in focal cerebral ischemic injury (FCII) after middle cerebral artery occlusion was studied using tPA gene-deficient (KO) mice and their wild-type (WT) littermates. The middle cerebral artery was occluded by thrombi induced by three different intensities of photochemical damage, a method that was newly introduced in mice. In both WT and KO mice, the intensity-dependent increase of FCII size was observed. The FCII size in tPA WT mice was smaller than in KO mice in cases of mild damage, whereas the FCII size was larger in WT mice than in KO mice in cases of severe damage. There was no difference in FCII size between WT and KO mice in cases of moderate damage. The number of microthrombi also increased with damage intensity in both WT and KO mice, but was less in WT mice at all intensities of damage. The results support the validity of the model of thrombotic occlusion by photochemical damage in mice, and suggest that endogenous tPA protects FCII through thrombolytic action on transient occlusion of middle cerebral artery with mild damage, but deteriorates on persistent occlusion with severe damage.

The activity of thrombinogenesis process is shown by thrombin restrain reaction by its physiological inhibitor antithrombin III, as a result of which biologically nonactive thrombinantithrombin III complexes (TAT) are created. The aim of the study was to evaluate TAT complexes in the blood of ischaemic stroke patients. 25 persons with ischaemic stroke and 15 controls took part in the examination. Taking into consideration brain CT outcomes 2 groups of patients were selected: with ischaemic stroke of small and great extent. The patients were also divided into a group of patients with less severe and with more severe ischaemic stroke, and into a group of patients with ischaemic stroke with and without accompanying atrial fibrillation. TAT complexes were determined with an enzyme-linked immunosorbent assay method on days 1st, 5th, and 12th after ischaemic stroke onset. TAT complexes were significantly higher in patients with cerebral infarction than in controls, significantly higher in patients with ischaemic stroke of great extent compared to patients with ischaemic stroke of small extent, and significantly higher in patients with more severe cerebral infarction than in patients with less severe cerebral infarction. In patients with cerebral infarction and atrial fibrillation TAT complexes were significantly higher only on the 1st day of the disease compared to patients with cerebral infarction without atrial fibrillation. Our investigations confirm intense activity of thrombinogenesis process in ischaemic stroke patients.

Apoptosis-related cell death is linked to oxidative stress and caspases in experimental cerebral ischemia. However, the role of oxidative stress in caspase activation and subsequent apoptotic cell death after cerebral ischemia is unknown. The authors evaluated the role of oxidative stress in ischemic cerebral infarction after photothrombosis and the relation between oxidative stress and caspase-related cell death 6 and 24 hours after ischemia with and without U-74389G, a potent free radical scavenger (10 mg/kg, 30 minutes before and after ischemia induction). Reactive oxygen species, detected by hydroethidine oxidation, and cytosolic cytochrome c were detected in early ischemic lesions. Western blot analysis showed the cleaved form and the increased level of the proform of caspase-3 in the ischemic lesion 24 hours after ischemia. Decreased caspase-3 immunoreactivity was detected in the antioxidant-treated group after ischemia. Decreased DNA fragmentation and laddering were detected and the lesion was smaller in the treated group after ischemia compared with the untreated group. Oxidative stress and cytochrome c release occur in the ischemic lesion after photothrombotic ischemia. The free radical scavenger attenuated caspase-3 up-regulation, DNA fragmentation, and the final lesion. The authors concluded that oxidative stress may mediate caspase-related apoptotic cell death and subsequent cortical infarction after photothrombotic ischemia.

Inflammatory mediators are involved in the pathogenesis of focal ischemic brain damage. In this study we used quantitative reverse transcriptase-polymerase chain reaction to analyze the spatiotemporal pattern of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and inducible nitric oxide synthase (iNOS) expression in focal ischemia of the rat brain. Focal ischemia of the rat parietal cortex was induced noninvasively by photothrombosis of cortical microvessels. In a proportion of the animals NMDA receptor signaling was blocked by the noncompetitive receptor antagonist MK-801. Within 4 h after ischemia we found induction of TNF-alpha and IL-1beta mRNA not only in the infarcts but also in all representative tissue samples removed from noninfarcted frontal, lateral, and occipital cortex of the ipsilateral, but not contralateral hemisphere. Contrastingly, the expression of iNOS mRNA remained restricted to the evolving infarcts. Pretreatment with MK-801 strongly inhibited remote cytokine expression (mean reduction by 80% relative to vehicle treated animals at 4 h; P<0.001) whereas in the lesions only partial reductions in the expression of IL-1beta and iNOS mRNA were found. Our data for the first time demonstrate remote cytokine induction following focal brain ischemia and suggest that NMDA receptor-mediated signaling can activate inflammatory gene expression independently from the occurrence of neuronal cell death.

1. The aim of the present study was to investigate the effects of a platelet-activating factor (PAF) antagonist (ginkgolide B; GB) on brain oedema, ion homeostasis, monoamine neurotransmitter disturbances in ischaemic penumbra and the neuroprotective mechanisms of the PAF antagonist. 2. Photochemically induced thrombotic cerebral ischaemia in tree shrews was used as the experimental model and changes in histopathology and the content of monoamine neurotransmitters (measured by a spectrofluorometer), water content (measured by a specific gravimetric method), sodium and calcium (measured by an atomic absorption spectrophotometer) in the penumbra were observed. 3. Results show that content of noradrenaline (NA), dopamine (DA) and 5-hydroxytryptamine (5-HT) decreased in the penumbra, while 5-HT metabolite 5-hydroxyindole-3-acetic acid, sodium, calcium and water contents increased markedly (P < 0.01) after photochemical reaction. 4. In tree shrews treated with GB (5 mg/kg, i.v.) 6 h after photochemical reaction, cortical NA, DA and 5-HT contents recovered to control levels and water and calcium contents decreased significantly (P < 0.01). 5. The results suggest that PAF may play an important role in inducing calcium overload, brain oedema and secondary brain damage in penumbra and that GB produces its neuroprotective effects by inhibiting the pathological manifestation of PAF.