UDP-Glucose/P2Y14 Receptor Signaling Exacerbates Neuronal Apoptosis After Subarachnoid Hemorrhage in Rats.

BACKGROUND: Subarachnoid hemorrhage (SAH) is a severe subtype of stroke with poor outcomes. Abnormal glucose metabolism often occurs after SAH, but the strict control of blood glucose levels is not always beneficial. This study aimed to investigate the contribution of uridine diphosphate glucose (UDP-G), an intermediate of glucose/glycogen metabolism, and its receptor P2Y14 (P2Y purinoceptor 14) to SAH pathology and explored the potential targeted treatments in rats. METHODS: A total of 218 Sprague-Dawley male rats were used. SAH was induced by endovascular perforation. Brain expressions of P2Y14, uridine diphosphate glucose (UDP-G), and its converting enzyme UGP2 (UDP-G pyrophosphorylase-2) were evaluated. Exogenous UDP-G or selective P2Y14 inhibitor was administered intranasally at 1 hour after SAH to explore their potential effects. Intranasal Ugp2 or P2ry14 siRNA was delivered 24 hours before SAH for mechanistic evaluation. Primary neuron culture and hemoglobin stimulation were used as in vitro model of SAH. Post-SAH evaluation included liquid chromatography-mass spectrometry measurement of brain endogenous UDP-G level, neurobehavioral assessments, Western blotting, immunohistochemistry, TUNEL staining, and Nissl staining. RESULTS: There was an acute elevation of endogenous brain UDP-G and UGP2 after SAH, and P2Y14 was expressed in neurons. Although P2Y14 inhibitor decreased neurological dysfunction, neuronal apoptosis, and proapoptotic molecules, exogenous UDP-G exacerbated these outcomes at 24 hours after SAH. Early inhibition of P2Y14 preserved long-term neuronal survival in the hippocampus, amygdala, and cortex with improved neurocognition and depressive-like behavior. In addition, in vivo knockdown of Ugp2- and P2ry14-reduced neurological deficits and proapoptotic molecules at 24 hours after SAH, and furthermore in vitro knockdown of P2ry14-reduced apoptosis in hemoglobin stimulated primary neuron. CONCLUSIONS: These findings suggest a detrimental role of brain UDP-G/P2Y14 signaling in SAH, as a part of glucose metabolic pathology at the tissue level. P2Y14 inhibitor 4-[4-(4-piperidinyl)phenyl]-7-[4-(trifluoromethyl)phenyl]-2-naphthalenecarboxylic acid hydrochloride may serve as a potential therapeutic target in treating patients with SAH.

Anti-Apoptotic Effects of AMPA Receptor Antagonist Perampanel in Early Brain Injury After Subarachnoid Hemorrhage in Mice.

This study was aimed to investigate if acute neuronal apoptosis is induced by activation of AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionate) receptors (AMPARs) and inhibited by a clinically available selective AMPAR antagonist and antiepileptic drug perampanel (PER) in subarachnoid hemorrhage (SAH), and if the mechanisms include upregulation of an inflammation-related matricellular protein periostin. Sham-operated and endovascular perforation SAH mice randomly received an administration of 3 mg/kg PER or the vehicle intraperitoneally. Post-SAH neurological impairments and increased caspase-dependent neuronal apoptosis were associated with activation of AMPAR subunits GluA1 and GluA2, and upregulation of periostin and proinflammatory cytokines interleukins-1ß and -6, all of which were suppressed by PER. PER also inhibited post-SAH convulsion-unrelated increases in the total spectral power on video electroencephalogram (EEG) monitoring. Intracerebroventricularly injected recombinant periostin blocked PER's anti-apoptotic effects on neurons. An intracerebroventricular injection of a selective agonist for GluA1 and GluA2 aggravated neurological impairment, neuronal apoptosis as well as periostin upregulation, but did not increase the EEG total spectral power after SAH. A higher dosage (10 mg/kg) of PER had even more anti-apoptotic effects compared with 3 mg/kg PER. Thus, this study first showed that AMPAR activation causes post-SAH neuronal apoptosis at least partly via periostin upregulation. A clinically available AMPAR antagonist PER appears to be neuroprotective against post-SAH early brain injury through the anti-inflammatory and anti-apoptotic effects, independent of the antiepileptic action, and deserves further study.

Fractalkine Enhances Hematoma Resolution and Improves Neurological Function via CX3CR1/AMPK/PPARγ Pathway After GMH.

BACKGROUND: Hematoma clearance has been a proposed therapeutic strategy for hemorrhagic stroke. This study investigated the impact of CX3CR1 (CX3C chemokine receptor 1) activation mediated by r-FKN (recombinant fractalkine) on hematoma resolution, neuroinflammation, and the underlying mechanisms involving AMPK (AMP-activated protein kinase)/PPARγ (peroxisome proliferator-activated receptor gamma) pathway after experimental germinal matrix hemorrhage (GMH). METHODS: A total of 313 postnatal day 7 Sprague Dawley rat pups were used. GMH was induced using bacterial collagenase by a stereotactically guided infusion. r-FKN was administered intranasally at 1, 25, and 49 hours after GMH for short-term neurological evaluation. Long-term neurobehavioral tests (water maze, rotarod, and foot-fault test) were performed 24 to 28 days after GMH with the treatment of r-FKN once daily for 7 days. To elucidate the underlying mechanism, CX3CR1 CRISPR, or selective CX3CR1 inhibitor AZD8797, was administered intracerebroventricularly 24 hours preinduction of GMH. Selective inhibition of AMPK/PPARγ signaling in microglia via intracerebroventricularly delivery of liposome-encapsulated specific AMPK (Lipo-Dorsomorphin), PPARγ (Lipo-GW9662) inhibitor. Western blot, Immunofluorescence staining, Nissl staining, Hemoglobin assay, and ELISA assay were performed. RESULTS: The brain expression of FKN and CX3CR1 were elevated after GMH. FKN was expressed on both neurons and microglia, whereas CX3CR1 was mainly expressed on microglia after GMH. Intranasal administration of r-FKN improved the short- and long-term neurobehavioral deficits and promoted M2 microglia polarization, thereby attenuating neuroinflammation and enhancing hematoma clearance, which was accompanied by an increased ratio of p-AMPK (phosphorylation of AMPK)/AMPK, Nrf2 (nuclear factor erythroid 2-related factor 2), PPARγ, CD36 (cluster of differentiation 36), CD163 (hemoglobin scavenger receptor), CD206 (the mannose receptor), and IL (interleukin)-10 expression, and decreased CD68 (cluster of differentiation 68), IL-1ß, and TNF (tumor necrosis factor) α expression. The administration of CX3CR1 CRISPR or CX3CR1 inhibitor (AZD8797) abolished the protective effect of FKN. Furthermore, selective inhibition of microglial AMPK/PPARγ signaling abrogated the anti-inflammation effects of r-FKN after GMH. CONCLUSIONS: CX3CR1 activation by r-FKN promoted hematoma resolution, attenuated neuroinflammation, and neurological deficits partially through the AMPK/PPARγ signaling pathway, which promoted M1/M2 microglial polarization. Activating CX3CR1 by r-FKN may provide a promising therapeutic approach for treating patients with GMH.

Epidermal Growth Factor Receptor Mediates Neuronal Apoptosis After Subarachnoid Hemorrhage in Mice.

BACKGROUND: Early brain injury including neuronal apoptosis is a main contributor to neurological deterioration after subarachnoid hemorrhage (SAH). This study was aimed to investigate whether EGFR (epidermal growth factor receptor)/NFκB (nuclear factor-kappa B) inducing kinase (NIK)/NFκB (p65 and p50) pathway is involved in the neuronal apoptosis after SAH in mice. METHODS: C57BL/6 adult male mice underwent endovascular perforation SAH modeling or sham-operation (n=286), and 86 mild SAH mice were excluded. In experiment 1, vehicle or an EGFR inhibitor (632.0 ng AG1478) was administered intraventricularly at 30 minutes postmodeling. At 24 or 72 hours, after neurological score was tested, brain water content, double immunolabeling with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and a neuronal marker antimicrotubule-associated protein-2 antibody, Western blotting using whole tissue lysate or nuclear protein extraction of the left cortex, and immunohistochemistry for cleaved caspase-3, phosphorylated (p-) EGFR, NIK, p-NFκB p65, and NFκB p105/50 were evaluated. In experiment 2, after sham or SAH modeling, AG1478+vehicle or AG1478+4.0 ng EGF was administered intraventricularly. The brain was used for TUNEL staining and immunohistochemistry after 24-hour observation. RESULTS: SAH group showed deteriorated neurological score (P<0.01, Mann-Whitney U test), more TUNEL- and cleaved caspase-3-positive neurons (P<0.01, ANOVA), and higher brain water content (P<0.01, Mann-Whitney U test), and these observations were improved in SAH-AG1478 group. Western blotting showed that expression levels of p-EGFR, p-p65, p50, and nuclear-NIK were increased after SAH (P<0.05, ANOVA), and decreased by AG1478 administration. Immunohistochemistry revealed these molecules localized in degenerating neurons. EGF administration resulted in neurological deterioration, increased TUNEL-positive neurons, and activation of EGFR, NIK, and NFκB. CONCLUSIONS: Activated EGFR, nuclear-NIK, and NFκB expressions were observed in cortical degenerating neurons after SAH, and were decreased by administration of AG1478, associated with suppression of TUNEL- and cleaved caspase-3-positive neurons. EGFR/NIK/NFκB pathway is suggested to be involved in neuronal apoptosis after SAH in mice.

Role of Estrogen-Related Receptor γ and PGC-1α/SIRT3 Pathway in Early Brain Injury After Subarachnoid Hemorrhage.

Estrogen-related receptors (ERRs) were shown to play an important role in the regulation of free radical-mediated pathology. This study aimed to investigate the neuroprotective effect of ERRγ activation against early brain injury (EBI) after subarachnoid hemorrhage (SAH) and the potential underlying mechanisms. In a rat model of SAH, the time course of ERRs and SIRT3 and the effects of ERRγ activation were investigated. ERRγ agonist DY131, selective inhibitor GSK5182, or SIRT3 selective inhibitor 3-TYP were administered intracerebroventricularly (icv) in the rat model of SAH. The use of 3-TYP was for validating SIRT3 as the downstream signaling of ERRγ activation. Post-SAH assessments included SAH grade, neurological score, Western blot, Nissl staining, and immunofluorescence staining in rats. In an vitro study, the ERRγ agonist DY131 and ERRγ siRNA were administered to primary cortical neurons stimulated by Hb, after which cell viability and neuronal deaths were accessed. Lastly, the brain ERRγ levels and neuronal death were accessed in SAH patients. We found that brain ERRγ expressions were significantly increased, but the expression of SIRT3 dramatically decreased after SAH in rats. In the brains of SAH rats, ERRγ was expressed primarily in neurons, astrocytes, and microglia. The activation of ERRγ with DY131 significantly improved the short-term and long-term neurological deficits, accompanied by reductions in oxidative stress and neuronal apoptosis at 24 h after SAH in rats. DY131 treatment significantly increased the expressions of PGC-1α, SIRT3, and Bcl-2 while downregulating the expressions of 4-HNE and Bax. ERRγ antagonist GSK5182 and SIRT3 inhibitor 3-TYP abolished the neuroprotective effects of ERRγ activation in the SAH rats. An in vitro study showed that Hb stimulation significantly increased intracellular oxidative stress in primary cortical neurons, and DY131 reduced such elevations. Primary cortical neurons transfected with the ERRγ siRNA exhibited notable apoptosis and abolished the protective effect of DY131. The examination of SAH patients' brain samples revealed increases in ERRγ expressions and neuronal apoptosis marker CC3. We concluded that ERRγ activation with DY131 ameliorated oxidative stress and neuronal apoptosis after the experimental SAH. The effects were, at least in part, through the ERRγ/PGC-1α/SIRT3 signaling pathway. ERRγ may serve as a novel therapeutic target to ameliorate EBI after SAH.

Roles of glutamate in brain injuries after subarachnoid hemorrhage.

Aneurysmal subarachnoid hemorrhage (SAH) is a stroke type with a high rate of mortality and morbidity. Post-SAH brain injury as a determinant of poor outcome is classified into the following two types: early brain injury (EBI) and delayed cerebral ischemia (DCI). EBI consists of various acute brain pathophysiologies that occur within the first 72 hours of SAH in a clinical setting. The underlying mechanisms of DCI are considered to be cerebral vasospasm or microcirculatory disturbance, which develops mostly 4 to 14 days after clinical SAH. Glutamate is the principal neurotransmitter in the central nervous system, but excessive glutamate is known to induce neurotoxicity. Experimental and clinical studies have revealed that excessive glutamates are released after SAH. In addition, many studies have reported the relationships between excessive glutamate release or overactivation of glutamate receptors and excitotoxicity, cortical spreading depolarization, seizure, increased blood-brain barrier permeability, neuroinflammation, microthrombosis formation, microvasospasm, cerebral vasospasm, impairments of brain metabolic supply and demand, impaired neurovascular coupling, and so on, all of which potentially contribute to the development of EBI or DCI. As glutamates always exert their functions through one or more of 4 major receptors of glutamates, it would be valuable to know the mechanisms as to how glutamates cause these pathologies, and the possibility that a glutamate receptor antagonist may block the pathologies. To prevent the mechanistic steps leading to glutamate-mediated neurotoxicity may ameliorate SAH-induced brain injuries and improve the outcomes. This review addresses the current knowledge of glutamate-mediated neurotoxicity, focusing on EBI and DCI after SAH.

Inhibition of AMPA (α-Amino-3-Hydroxy-5-Methyl-4-Isoxazole Propionate) Receptor Reduces Acute Blood-Brain Barrier Disruption After Subarachnoid Hemorrhage in Mice.

Activation of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor (AMPAR) is thought to cause acute brain injury, but the role remains poorly understood in subarachnoid hemorrhage (SAH). This study was conducted to evaluate if AMPAR activation induces acute blood-brain barrier (BBB) disruption after SAH. C57BL/6 male adult mice (n = 117) underwent sham or filament perforation SAH modeling, followed by a random intraperitoneal injection of vehicle or two dosages (1 mg/kg or 3 mg/kg) of a selective noncompetitive AMPAR antagonist perampanel (PER) at 30 min post-modeling. The effects were evaluated by mortality, neurological scores, and brain water content at 24-48 h and video electroencephalogram monitoring, immunostaining, and Western blotting at 24 h post-SAH. PER significantly suppressed post-SAH neurological impairments, brain edema, and BBB disruption. SAH developed epileptiform spikes without obvious convulsion, which were also inhibited by PER. Western blotting showed that the expression of AMPAR subunits GluA1 and GluA2 was unchanged after SAH, but they were significantly activated after SAH. PER prevented post-SAH activation of GluA1/2, associated with the suppression of post-SAH induction of tenascin-C, a causative mediator of post-SAH BBB disruption. Meanwhile, an intracerebroventricular injection of a subtype-selective GluA1/2 agonist augmented the activation of GluA1/2 and the induction of tenascin-C in brain capillary endothelial cells and aggravated post-SAH BBB disruption without increases in epileptiform spikes. Neurological impairments and brain edema were not correlated with the occurrence of epileptiform spikes. This study first showed that AMPAR plays an important role in the development of post-SAH BBB disruption and can be a novel therapeutic target against it.

Nonfasting Triglyceride as an Independent Predictor of Carotid Restenosis After Carotid Endarterectomy or Carotid Artery Stenting.

OBJECTIVE: Nonfasting serum triglyceride (TG) level is attracting more and more attention as an atherosclerosis-promoting factor. However, no study has investigated the relationships between nonfasting TG levels and carotid restenosis after carotid endarterectomy (CEA) or carotid artery stenting (CAS). This study was conducted to investigate if nonfasting TG levels can be used to assess a risk for carotid restenosis after CEA or CAS. METHODS: This was a single-center retrospective study. We reviewed 201 consecutive primary carotid artery revascularization procedures (39 CEAs and 162 CASs), which were performed from 2008 to 2018 for 179 patients (163 men and 16 women) with atherosclerotic carotid stenosis, and were followed up for at least 1 year. Clinical variables including nonfasting lipid profiles and findings of magnetic resonance plaque imaging were compared between groups with and without postprocedural carotid restenosis (≥50% stenosis on ultrasonography). RESULTS: During a mean follow-up period of 1413 days, 24 of 201 carotid stenosis procedures (11.9%) suffered restenosis after successful revascularization procedures. Multivariate analyses demonstrated that nonfasting TG level was the only independent risk factor of postprocedural restenosis. The receiver operating characteristic curve analyses revealed that a cutoff value of nonfasting TG to discriminate postprocedural carotid restenosis was 127.5 mg/dL, which was much lower than the upper limit of normal. CONCLUSIONS: This study showed that nonfasting TG level may be a useful marker to predict carotid restenosis after CEA or CAS, and could be a new therapeutic target to prevent carotid restenosis after revascularization procedures.

Higher Non-fasting Serum Triglyceride Preceding the Carotid Stenosis Progression.

The present study was conducted to investigate whether non-fasting serum triglyceride (TG) levels can be used to assess a risk for the progression of carotid artery stenosis. This was a single-center retrospective study. Consecutive 96 patients with ≥50% stenosis of at least unilateral cervical internal carotid artery and normal fasting serum low-density lipoprotein cholesterol (LDL-C) levels of ≤140 mg/dL were followed up for at least 1 year (mean, 3.1 years), and clinical variables were compared between patients with and without carotid stenosis progression (≥10% increases in the degree on ultrasonography). Carotid stenosis progression was shown in 21 patients, associated with less frequent treatment with calcium channel blockers (CCBs), higher non-fasting TG and glucose levels. In carotid artery-based analyses including <50% stenosis side, stenosis progression was shown in 23 of 121 arteries except for those with complete occlusion and less than 1-year follow-up period because of carotid artery stenting (CAS) or carotid endarterectomy (CEA). Stenosis progression was more frequently observed in symptomatic and/or radiation-induced lesions, and was also accompanied with less frequent treatment with CCBs, higher non-fasting TG and glucose levels in carotid artery-based analyses. The receiver operating characteristic (ROC) curve analyses revealed that a cutoff value of non-fasting TG to discriminate carotid stenosis progression was 169.5 mg/dL for carotid arteries with the baseline stenosis of <50%, and 154.5mg/dL for those of ≥50%. Non-fasting TG level was an independent risk factor of carotid stenosis progression, and more strict control of non-fasting TG may be necessary for higher degree of carotid artery stenosis.

Clarithromycin Ameliorates Early Brain Injury After Subarachnoid Hemorrhage via Suppressing Periostin-Related Pathways in Mice.

Subarachnoid hemorrhage (SAH) remains a life-threatening disease, and early brain injury (EBI) is an important cause of poor outcomes. The authors have reported that periostin, a matricellular protein, is one of key factors of post-SAH EBI. Clarithromycin (CAM) is a worldwide antibiotic that can inhibit periostin expression. This study aimed to investigate whether CAM suppressed EBI after experimental SAH, focusing on blood-brain barrier (BBB) disruption, an important pathology of EBI. C57BL/6 male adult mice underwent endovascular perforation SAH modeling (n = 139) or sham operation (n = 30). Different dosages (25, 50, or 100 mg/kg) of CAM or the vehicle (n = 16, 52, 13, and 58, respectively) were randomly administered by an intramuscular injection 5 min after SAH induction. Post-SAH 50 mg/kg CAM treatment most effectively improved neurological scores and brain water content at 24 and 48 h and reduced immunoglobulin G extravasation at 24 h compared with vehicle-treated SAH mice (p < 0.01). Western blotting showed that post-SAH BBB disruption was associated with increased expressions of periostin, phosphorylated signal transducer and activator of transcription 1 and 3, matrix metalloproteinase-9, and the consequent degradation of zonula occludens-1, which were suppressed by 50 mg/kg CAM treatment (p < 0.05, respectively, versus vehicle-treated SAH mice). Periostin and its related molecules were upregulated in capillary endothelial cells and neurons after SAH. An intracerebroventricular injection of recombinant periostin blocked the neuroprotective effects of CAM in SAH mice (n = 6, respectively; p < 0.05). In conclusion, this study first demonstrated that CAM improved post-SAH EBI in terms of BBB disruption at least partly via the suppression of periostin-related pathways.

Higher Plasma Osteopontin Concentrations Associated with Subsequent Development of Chronic Shunt-Dependent Hydrocephalus After Aneurysmal Subarachnoid Hemorrhage.

A matricellular protein osteopontin (OPN) is considered to exert neuroprotective and healing effects on neurovascular injuries in an acute phase of aneurysmal subarachnoid hemorrhage (SAH). However, the relationships between OPN expression and chronic shunt-dependent hydrocephalus (SDHC) have never been investigated. In 166 SAH patients (derivation and validation cohorts, 110 and 56, respectively), plasma OPN levels were serially measured at days1-3, 4-6, 7-9, and 10-12 after aneurysmal obliteration. The OPN levels and clinical factors were compared between patients with and without subsequent development of chronic SDHC. Plasma OPN levels in the SDHC patients increased from days 1-3 to days 4-6 and remained high thereafter, while those in the non-SDHC patients peaked at days 4-6 and then decreased over time. Plasma OPN levels had no correlation with serum levels of C-reactive protein (CRP), a systemic inflammatory marker. Univariate analyses showed that age, modified Fisher grade, acute hydrocephalus, cerebrospinal fluid drainage, and OPN and CRP levels at days 10-12 were significantly different between patients with and without SDHC. Multivariate analyses revealed that higher plasma OPN levels at days 10-12 were an independent factor associated with the development of SDHC, in addition to a more frequent use of cerebrospinal fluid drainage and higher modified Fisher grade at admission. Plasma OPN levels at days 10-12 maintained similar discrimination power in the validation cohort and had good calibration on the Hosmer-Lemeshow goodness-of-fit test. Prolonged higher expression of OPN may contribute to the development of post-SAH SDHC, possibly by excessive repairing effects promoting fibrosis in the subarachnoid space.

Cerebrovascular pathophysiology of delayed cerebral ischemia after aneurysmal subarachnoid hemorrhage.

Aneurysmal subarachnoid hemorrhage (SAH) remains a serious cerebrovascular disease. Even if SAH patients survive the initial insults, delayed cerebral ischemia (DCI) may occur at 4 days or later post-SAH. DCI is characteristics of SAH, and is considered to develop by blood breakdown products and inflammatory reactions, or secondary to early brain injury, acute pathophysiological events that occur in the brain within the first 72 hours of aneurysmal SAH. The pathology underlying DCI may involve large artery vasospasm and/or microcirculatory disturbances by microvasospasm, microthrombosis, dysfunction of venous outflow and compression of microvasculature by vasogenic or cytotoxic tissue edema. Recent clinical evidence has shown that large artery vasospasm is not the only cause of DCI, and that both large artery vasospasm-dependent and -independent cerebral infarction causes poor outcome. Animal studies suggest that mechanisms of vasospasm may differ between large artery and arterioles or capillaries, and that many kinds of cells in the vascular wall and brain parenchyma may be involved in the pathogenesis of microcirculatory disturbances. The impairment of the paravascular and glymphatic systems also may play important roles in the development of DCI. As pathological mediators for DCI, glutamate and several matricellular proteins have been investigated in addition to inflammatory molecules. Glutamate is involved in excitotoxicity contributing to cortical spreading ischemia and epileptic activity-related events. Microvascular dysfunction is an attractive mechanism to explain the cause of poor outcomes independently of large cerebral artery vasospasm, but needs more studies to clarify the pathophysiologies or mechanisms and to develop a novel therapeutic strategy.

Prognostic factors varying with age in patients with aneurysmal subarachnoid hemorrhage.

With the advent of an aging society, more elderly patients with aneurysmal subarachnoid hemorrhage (aSAH) have been treated. We investigated if prognostic factors differ with age in aSAH patients. In a prospectively maintained aSAH database at multiple institutions from 2013 to 2016, 238 patients who underwent clipping or coiling for a ruptured aneurysm within 48 h of onset were divided into elderly (≥75 years; 57 patients) and non-elderly groups, or categorized into 4-age groups (<54, 55-64, 65-74, and ≥75 years). Prognostic factors and clinical characteristics were retrospectively analyzed. The elderly group had a higher incidence of pre-morbidities, co-morbidities, poor admission World Federation of Neurological Surgeons (WFNS) grades, modified Fisher grade 4, and resultantly 90-day poor outcomes (modified Rankin scale [mRS] 3-6). Multivariate logistic regression analyses revealed that independent determinants for poor outcomes were hypertension and modified Fisher grade 4 in the elderly group, and admission WFNS grades IV-V, systemic complications, non-procedural cerebral infarction and shunt-dependent chronic hydrocephalus in the non-elderly group. The 4-age group analyses showed that higher age group was more frequently associated with the prognostic factors. As higher age itself causes poor outcomes and more association of prognostic factors, prognostic factors in elderly patients may be rather limited.

Tenascin-C in brain injuries and edema after subarachnoid hemorrhage: Findings from basic and clinical studies.

Subarachnoid hemorrhage (SAH) by a rupture of cerebral aneurysms remains the most devastating cerebrovascular disease. Early brain injury (EBI) is increasingly recognized to be the primary determinant for poor outcomes, and also considered to cause delayed cerebral ischemia (DCI) after SAH. Both clinical and experimental literatures emphasize the impact of global cerebral edema in EBI as negative prognostic and direct pathological factors. The nature of the global cerebral edema is a mixture of cytotoxic and vasogenic edema, both of which may be caused by post-SAH induction of tenascin-C (TNC) that is an inducible, non-structural, secreted and multifunctional matricellular protein. Experimental SAH induces TNC in brain parenchyma in rats and mice. TNC knockout suppressed EBI in terms of brain edema, blood-brain barrier disruption, neuronal apoptosis and neuroinflammation, associated with the inhibition of post-SAH activation of mitogen-activated protein kinases and nuclear factor-kappa B in mice. In a clinical setting, more severe SAH increases more TNC in cerebrospinal fluid and peripheral blood, which could be a surrogate marker of EBI and predict DCI development and outcomes. In addition, cilostazol, a selective inhibitor of phosphodiesterase type III that is a clinically available anti-platelet agent and is known to suppress TNC induction, dose-dependently inhibited delayed cerebral infarction and improved outcomes in a pilot clinical study. Thus, further studies may facilitate application of TNC as biomarkers for non-invasive diagnosis or assessment of EBI and DCI, and lead to development of a molecular target drug against TNC, contributing to the improvement of post-SAH outcomes.

Morphological Characteristics of Neuronal Death After Experimental Subarachnoid Hemorrhage in Mice Using Double Immunoenzymatic Technique.

Subarachnoid hemorrhage (SAH) is a devastating disease. Neuronal death is an important pathophysiology in the acute phase of SAH, but the histopathological features of dying neurons have been poorly studied. Using several staining methods including terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and microtubule-associated protein 2 (MAP-2) double immunolabeling, we investigated the morphological changes of nucleus and cytoskeleton in neurons and sought susceptible areas to neuronal death in filament perforation SAH mice under light microscope. TUNEL and MAP-2 double immunolabeling clearly showed morphological features of shrunken cytoplasm and sometimes curl-like fibers in dying neurons, besides nuclear abnormalities. More dying neurons were detected in the moderate SAH group than in the mild SAH group, and the temporal base cortex was the most susceptible area to neuronal death with deoxyribonucleic acid (DNA) damage among the cerebral cortices and hippocampus at 24 hr after SAH (p<0.01, ANOVA). Lesser hippocampal neuronal death was observed at 24 hr, but neuronal death was significantly increased in the CA1 region at 7 days after SAH (p<0.05, unpaired t-test). Using TUNEL and MAP-2 double immunolabeling, morphological features of not only the nucleus but also the cytoplasm in post-SAH neuronal death with DNA damage can be observed in detail under light microscope.

Potential roles of matricellular proteins in stroke.

Both ischemic and hemorrhagic strokes are still serious diseases with high mortalities and morbidities. To improve outcomes of strokes, new therapeutic approaches need to be developed. Matricellular proteins are inducible, multifunctional and non-structural extracellular matrix proteins, which are definitely differentiated from classical ones due to the unusual diversity of functions. There are many matricellular proteins known, most of which may be involved in the pathophysiology and the protective or repairing mechanisms of strokes. This article reviews the available information regarding potential roles of matricellular proteins in stroke, and discusses the potential therapeutic approaches against stroke using matricellular proteins.

Machine Learning Analysis of Matricellular Proteins and Clinical Variables for Early Prediction of Delayed Cerebral Ischemia After Aneurysmal Subarachnoid Hemorrhage.

Although delayed cerebral ischemia (DCI) is a well-known complication after subarachnoid hemorrhage (SAH), there are no reliable biomarkers to predict DCI development. Matricellular proteins (MCPs) have been reported relevant to DCI and expected to become biomarkers. As machine learning (ML) enables the classification of various input data and the result prediction, the aim of this study was to construct early prediction models of DCI development with clinical variables and MCPs using ML analyses. Early-stage clinical data of 95 SAH patients in a prospective cohort were analyzed and applied to a ML algorithm, random forest, to construct three prediction models: (1) a model with only clinical variables on admission, (2) a model with only plasma levels of MCP (periostin, osteopontin, and galectin-3) at post-onset days 1-3, and (3) a model with both clinical variables on admission and MCP values at days 1-3. The prediction accuracy of the development of DCI, angiographic vasospasm, or cerebral infarction and the importance of each feature were computed. The prediction accuracy of DCI development was 93.9% in model 1, 87.2% in model 2, and 95.1% in model 3, but that of angiographic vasospasm or cerebral infarction was lower. The three most important features in model 3 for DCI were periostin, osteopontin, and galectin-3, followed by aneurysm location. All of the early-stage prediction models of DCI development constructed by ML worked with high accuracy and sensitivity. One-time early-stage measurement of plasma MCPs served for reliable prediction of DCI development, suggesting their potential utility as biomarkers.

Potential therapeutic molecular targets for blood-brain barrier disruption after subarachnoid hemorrhage.

Aneurysmal subarachnoid hemorrhage remains serious hemorrhagic stroke with high morbidities and mortalities. Aneurysm rupture causes arterial bleeding-induced mechanical brain tissue injuries and elevated intracranial pressure, followed by global cerebral ischemia. Post-subarachnoid hemorrhage ischemia, tissue injuries as well as extravasated blood components and the breakdown products activate microglia, astrocytes and Toll-like receptor 4, and disrupt blood-brain barrier associated with the induction of many inflammatory and other cascades. Once blood-brain barrier is disrupted, brain tissues are directly exposed to harmful blood contents and immune cells, which aggravate brain injuries furthermore. Blood-brain barrier disruption after subarachnoid hemorrhage may be developed by a variety of mechanisms including endothelial cell apoptosis and disruption of tight junction proteins. Many molecules and pathways have been reported to disrupt the blood-brain barrier after subarachnoid hemorrhage, but the exact mechanisms remain unclear. Multiple independent and/or interconnected signaling pathways may be involved in blood-brain barrier disruption after subarachnoid hemorrhage. This review provides recent understandings of the mechanisms and the potential therapeutic targets of blood-brain barrier disruption after subarachnoid hemorrhage.

Plasma Periostin and Delayed Cerebral Ischemia After Aneurysmal Subarachnoid Hemorrhage.

Delayed cerebral ischemia (DCI) is a serious complication of aneurysmal subarachnoid hemorrhage (SAH). Matricellular protein periostin (POSTN) has been found to be upregulated and linked with early brain injury after experimental SAH. The aim of the present study was to investigate the relationship between plasma POSTN levels and various clinical factors including serum levels of C-reactive protein (CRP), an inflammatory marker, in 109 consecutive SAH patients whose POSTN levels were measured at days 1-12 after aneurysmal obliteration. DCI developed in 16 patients associated with higher incidence of angiographic vasospasm, cerebral infarction, and 90-day worse outcomes. POSTN levels peaked at days 4-6 before DCI development. Cerebrospinal fluid (CSF) drainage was associated with reduced POSTN levels, but did not influence CRP levels. There was no correlation between POSTN levels and other treatments or CRP levels. To predict DCI development, receiver-operating characteristic curves indicated that the most reasonable cutoff POSTN levels were obtained at days 1-3 in patients without CSF drainage (80.5 ng/ml; specificity, 77.6%; sensitivity, 85.7%). Multivariate analyses using variables obtained by day 3 revealed that POSTN level was an independent predictor of DCI. POSTN levels over the cutoff value were associated with higher incidence of DCI, but not angiographic vasospasm. This study shows for the first time that CSF drainage may reduce plasma POSTN levels, and that POSTN levels may increase prior to the development of DCI with and without vasospasm irrespective of systemic inflammatory reactions in clinical settings. These findings suggest POSTN as a new therapeutic molecular target against post-SAH DCI.