Search

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.

Neuroelectric disruptions such as seizures and cortical spreading depolarization may contribute to the development of delayed cerebral ischemia (DCI) after aneurysmal subarachnoid hemorrhage (SAH). However, effects of antiepileptic drug prophylaxis on outcomes remain controversial in SAH. The authors investigated if prophylactic administration of new-generation antiepileptic drugs levetiracetam and perampanel was beneficial against delayed neurovascular events after SAH. This was a retrospective single-center cohort study of 121 consecutive SAH patients including 56 patients of admission World Federation of Neurological Surgeons grades IV−V who underwent aneurysmal obliteration within 72 hours post-SAH from 2013 to 2021. Prophylactic antiepileptic drugs differed depending on the study terms: none (2013−2015), levetiracetam for patients at high risks of seizures (2016−2019), and perampanel for all patients (2020−2021). The 3rd term had the lowest occurrence of delayed cerebral microinfarction on diffusion-weighted magnetic resonance imaging and the tendency of reduced DCI. Other outcome measures were similar among the 3 terms including incidences of angiographic vasospasm, computed tomography-detectable delayed cerebral infarction, seizures, and 3-month good outcomes (modified Rankin Scale 0−2). The present study suggests that prophylactic administration of levetiracetam and perampanel at least does not worsen outcomes, and that perampanel may have the potential to reduce DCI by preventing microcirculatory disturbances after SAH. Further studies are warranted to investigate anti-DCI effects of a selective α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor antagonist perampanel in SAH patients in a large-scale prospective study.

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

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.

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.

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

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.

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.

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.

Introduction: Tenascin-C (TNC) is one of pleiotropic matricellular proteins, which are non-structural and secreted extracellular matrix proteins that exert diverse functions. Recently we showed that TNC is involved in the mechanisms of cerebral vasospasm and early brain injury (EBI) after subarachnoid hemorrhage (SAH). However, the role of TNC has not been sufficiently investigated in SAH. Materials and Methods: First, TNC knockout (TNKO) mice SAH models were produced by endovascular perforation method, and examined the direct evidence that TNC is induced after SAH and causes cerebral vasospasm and EBI. Second, TNC was measured in serum from 156 aneurysmal SAH patients, and cilostazol (0-300 mg/day) was administered postoperatively, and investigated as to the dose-dependent effect of cilostazol on cerebral vasospasm, delayed cerebral ischemia and infarction. Results: In experimental SAH, TNKO prevented blood-brain barrier disruption and brain edema formation by inhibiting mitogen-activated protein kinase (MAPK)-mediated matrix metalloproteinase-9 activation. TNKO also suppressed post-SAH induction of another matricellular protein, periostin, which aggravates post-SAH EBI. TNKO also prevented neuroinflammation and neuronal apoptosis via inhibiting upregulation of Toll-like receptor 4, phosphorylation of nuclear factor-κB, and induction of proinflammatory cytokines in neurons. TNKO suppressed post-SAH cerebral vasospasm via inhibiting the activation of MAPKs. In the clinical study, cilostazol treatment suppressed plasma TNC levels from days 1-3 to days 10-12 post-SAH, and showed dose-dependent effects against delayed cerebral infarction, leading to improved outcome. Multivariate analyses revealed that 300 mg/day cilostazol treatment was an independent determinant against poor outcomes post-SAH. Conclusions: TNKO exerted protective effects against neuroinflammation, blood-brain barrier disruption, neuronal apoptosis, and cerebral vasospasm. The 300 mg/day cilostazol may improve post-SAH outcomes by reducing plasma TNC levels and delayed cerebral infarction. Further investigations may provide a novel therapeutic approach targeting TNC.

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.

There are no direct evidences showing the linkage between Toll-like receptor 4 (TLR4) and blood-brain barrier (BBB) disruption after subarachnoid hemorrhage (SAH). The purpose of this study was to examine if selective blockage of TLR4 prevents BBB disruption after SAH in mice and if the TLR4 signaling involves mitogen-activated protein kinases (MAPKs). One hundred and fifty-one C57BL/6 male mice underwent sham or endovascular perforation SAH operation, randomly followed by an intracerebroventricular infusion of vehicle or two dosages (117 or 585 ng) of a selective TLR4 antagonist IAXO-102 at 30 min post-operation. The effects were evaluated by survival rates, neurological scores, and brain water content at 24-72 h and immunoglobulin G immunostaining and Western blotting at 24 h post-SAH. IAXO-102 significantly prevented post-SAH neurological impairments, brain edema, and BBB disruption, resulting in improved survival rates. IAXO-102 also significantly suppressed post-SAH activation of a major isoform of MAPK p46 c-Jun N-terminal kinase (JNK) and matrix metalloproteinase-9 as well as periostin induction and preserved tight junction protein zona occludens-1. Another selective TLR4 antagonist TAK-242, which has a different binding site from IAXO-102, also showed similar effects to IAXO-102. This study first provided the evidence that TLR4 signaling is involved in post-SAH acute BBB disruption and that the signaling is mediated at least partly by JNK activation. TLR4-targeted therapy may be promising to reduce post-SAH morbidities and mortalities.

Bleeding from meningiomas is well known, but massive subdural hemorrhage from a very small meningioma is rare. A 61-year-old woman presented with a sudden-onset headache and slight right hemiparesis without a history of trauma. Computed tomographic scan showed bilateral acute/subacute interhemispheric subdural hematoma, but contrast-enhanced computed tomography (CT) scan, non-enhanced magnetic resonance imaging (MRI) and digital subtraction angiography failed to detect the cause. The hematoma was conservatively treated. Three weeks later, CT scans showed a vestige of the hematoma along the falx. However, repeated angiogram revealed a tumor stain on the falx supplied by the middle meningeal arteries, leading to the tentative diagnosis of meningioma. The tumor was removed and histologically diagnosed as angiomatous meningioma. It is rare that falx meningioma causes massive interhemispheric subdural hematoma, and the diagnosis of the causative lesion is challenging if tumor is small. We review the literature and discuss the characteristics.

Aneurysmal subarachnoid hemorrhage remains devastating, and the most important determinant of poor outcome is early brain injury (EBI). In clinical settings, as a surrogate marker of EBI, loss of consciousness at ictus, poor initial clinical grades, and some radiographic findings are used, but these markers are somewhat subjective. Thus, it is imperative to find biomarkers of EBI that have beneficial prognostic and therapeutic implications. In our opinion, an ideal biomarker is a molecule that is implicated in the pathogenesis of both EBI and subsequently developing delayed cerebral ischemia (DCI), being a therapeutic target, and can be measured easily in the peripheral blood in an acute stage. A good candidate of such a biomarker is a matricellular protein, which is a secreted, inducible and multifunctional extracellular matrix protein. There are many kinds of matricellular proteins reported, but only tenascin-C, osteopontin, galectin-3 and periostin are reported relevant to EBI and DCI. Reliable biomarkers of EBI may stratify aneurysmal subarachnoid hemorrhage patients into categories of risk to develop DCI, and allow objective monitoring of the response to treatment for EBI and earlier diagnosis of DCI. This review emphasizes that further investigation of matricellular proteins as an avenue for biomarker discovery is warranted.

A matricellular protein galectin-3 is involved in tissue injury and inflammation, but the role of galectin-3 remains unclear in aneurysmal subarachnoid hemorrhage (SAH). The purpose of this study was to assess whether acute-stage galectin-3 levels were associated with the subsequent development of neurovascular events and outcome after SAH. This study included 83 consecutive patients diagnosed with aneurysmal SAH of resuscitated World Federation of Neurological Surgeons (WFNS) grades 1-3. Plasma galectin-3 levels were once measured on days 1-3 (the day after clipping or coiling). Fifteen patients had poor outcomes, which were associated with increasing age, female, pre-onset morbidity, worse WFNS grade, modified Fisher computed tomography scale, acute hydrocephalus, and higher galectin-3 levels compared with good outcomes. Multivariate analyses revealed that plasma galectin-3 was an independent determinant for poor outcome (odds ratio, 3.08; 95% confidence interval, 1.58-6.00; p = 0.001). Among post-SAH neurovascular events occurring on day 4 and thereafter, delayed cerebral ischemia and infarction, but not angiographic vasospasm and shunt-dependent hydrocephalus, showed significantly higher plasma galectin-3 levels on days 1-3. The receiver operating characteristic curve indicated that plasma galectin-3 with a cutoff value of 3.30 or 3.48 ng/ml predicted delayed cerebral infarction development or poor outcome (specificity, 62.5%, 70.6%; sensitivity, 90.9%, 73.3%, respectively). The findings suggest that plasma galectin-3 levels on days 1-3 would be a useful biomarker for predicting subsequent development of delayed cerebral infarction and eventual poor outcome and provide a new candidate, which may mediate between post-SAH early brain injury or inflammation and delayed cerebral infarction without vasospasm.

Background and Purpose— A matricellular protein tenascin-C is implicated in early brain injury after experimental subarachnoid hemorrhage (SAH). This study first evaluated the role of another matricellular protein periostin and the relationships with tenascin-C in post-SAH early brain injury. Methods— Wild-type (n=226) and tenascin-C knockout (n=9) C57BL/6 male adult mice underwent sham or filament perforation SAH modeling. Vehicle, anti-periostin antibody, or recombinant periostin was randomly administrated by an intracerebroventricular injection at 30 minutes post-modeling. Neuroscores, SAH grading, brain water content, immunostaining, and Western blotting were blindly evaluated at 24 to 48 hours post-SAH. Results— Periostin was induced in brain capillary endothelial cells and neurons at 24 hours post-SAH. Anti-periostin antibody improved post-SAH neurobehavior, brain edema, and blood–brain barrier disruption associated with downregulation of tenascin-C, inactivation of p38, extracellular signal-related kinase 1/2 and matrix metalloproteinase-9, and subsequent preservation of zona occludens-1. Recombinant periostin aggravated post-SAH brain edema and tenascin-C induction. Tenascin-C knockout prevented post-SAH neurobehavioral impairments and periostin induction. Conclusions— Periostin may cause post-SAH early brain injury through activating downstream signaling pathways and interacting with tenascin-C, providing a novel approach for the treatment of early brain injury.

Toll-like receptor 4 (TLR4) is expressed in various cell types in the central nervous system and exerts maximal inflammatory responses among the TLR family members. TLR4 can be activated by many endogenous ligands having damage-associated molecular patterns including heme and fibrinogen at the rupture of a cerebral aneurysm, and therefore its activation is reasonable as an initial step of cascades to brain injuries after aneurysmal subarachnoid hemorrhage (SAH). TLR4 activation induces tenascin-C (TNC), a representative of matricellular proteins that are a class of inducible, nonstructural, secreted, and multifunctional extracellular matrix glycoproteins. TNC is also an endogenous activator and inducer of TLR4, forming positive feedback mechanisms leading to more activation of the signaling transduction. Our studies have demonstrated that TLR4 as well as TNC are involved in inflammatory reactions, blood-brain barrier disruption, neuronal apoptosis, and cerebral vasospasm after experimental SAH. This article reviews recent understanding of TLR4 and TNC in SAH to suggest that the TLR4-TNC signaling may be an important therapeutic target for post-SAH brain injuries.

Early brain injury is now considered as an important cause of delayed neurological deterioration after aneurysmal subarachnoid hemorrhage (SAH), and neuronal apoptosis is one of the constituents of early brain injury. Caspase family is popular proteases in apoptotic pathways, but there also exist caspase-independent cell death pathways in many pathologic states. In this study, we investigated the ratio of caspase-related and caspase-unrelated neuronal deaths in a mice endovascular perforation SAH model. At 24 h after SAH, about half of neurons in the perforation-side cortex showed increased cleaved caspase-3 immunoreactivity. On the other hand, about half of cleaved caspase-3-immunonegative neurons showed abnormal morphology, suggesting that they were in the process of some sort of cell death in the absence of caspase-3 activity. These findings suggest that both caspase-dependent and caspase-independent signaling pathways may cause neuronal death after SAH.

Vasospasm after subarachnoid hemorrhage (SAH) has been studied, but the mechanisms remain to be unveiled. Tenascin-C (TNC), which is a matricellular protein and reported to increase in spastic cerebral artery wall after SAH, is a ligand for both Toll-like receptor 4 (TLR4) and epidermal growth factor receptor (EGFR). Our previous studies suggested the involvement of TNC and these receptors in vasoconstriction or vasospasm after SAH. In this study, we investigated whether upregulation of TNC and TLR4 is observed and if an EGFR inhibitor has suppressive effects against them in a mice endovascular perforation SAH model. At 24 h after SAH, TNC and TLR4 expressions were widely observed in spastic cerebral arteries, and these expressions were suppressed by the administration of an EGFR inhibitor. From these results, EGFR inhibitors possibly suppress the expression of not only EGFR but also TLR4 at least partly through regulating TNC upregulation. More studies are needed to clarify the precise mechanisms linking these receptors.

Toll-like receptor 4 (TLR4) is expressed in various cell types in the central nervous system and exerts maximal inflammatory responses among the TLR family members. TLR4 can be activated by many endogenous ligands having damage-associated molecular patterns including heme and fibrinogen at the rupture of a cerebral aneurysm, and therefore its activation is reasonable as an initial step of cascades to brain injuries after aneurysmal subarachnoid hemorrhage (SAH). TLR4 activation induces tenascin-C (TNC), a representative of matricellular proteins that are a class of inducible, nonstructural, secreted, and multifunctional extracellular matrix glycoproteins. TNC is also an endogenous activator and inducer of TLR4, forming positive feedback mechanisms leading to more activation of the signaling transduction. Our studies have demonstrated that TLR4 as well as TNC are involved in inflammatory reactions, blood-brain barrier disruption, neuronal apoptosis, and cerebral vasospasm after experimental SAH. This article reviews recent understanding of TLR4 and TNC in SAH to suggest that the TLR4-TNC signaling may be an important therapeutic target for post-SAH brain injuries.

Vasospasm after subarachnoid hemorrhage (SAH) has been studied, but the mechanisms remain to be unveiled. Tenascin-C (TNC), which is a matricellular protein and reported to increase in spastic cerebral artery wall after SAH, is a ligand for both Toll-like receptor 4 (TLR4) and epidermal growth factor receptor (EGFR). Our previous studies suggested the involvement of TNC and these receptors in vasoconstriction or vasospasm after SAH. In this study, we investigated whether upregulation of TNC and TLR4 is observed and if an EGFR inhibitor has suppressive effects against them in a mice endovascular perforation SAH model. At 24 h after SAH, TNC and TLR4 expressions were widely observed in spastic cerebral arteries, and these expressions were suppressed by the administration of an EGFR inhibitor. From these results, EGFR inhibitors possibly suppress the expression of not only EGFR but also TLR4 at least partly through regulating TNC upregulation. More studies are needed to clarify the precise mechanisms linking these receptors.

Early brain injury is now considered as an important cause of delayed neurological deterioration after aneurysmal subarachnoid hemorrhage (SAH), and neuronal apoptosis is one of the constituents of early brain injury. Caspase family is popular proteases in apoptotic pathways, but there also exist caspase-independent cell death pathways in many pathologic states. In this study, we investigated the ratio of caspase-related and caspase-unrelated neuronal deaths in a mice endovascular perforation SAH model. At 24 h after SAH, about half of neurons in the perforation-side cortex showed increased cleaved caspase-3 immunoreactivity. On the other hand, about half of cleaved caspase-3-immunonegative neurons showed abnormal morphology, suggesting that they were in the process of some sort of cell death in the absence of caspase-3 activity. These findings suggest that both caspase-dependent and caspase-independent signaling pathways may cause neuronal death after SAH.

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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13311-018-00707-y) contains supplementary material, which is available to authorized users.

Background and Purpose— Plasma levels of galectin-3—a matricellular protein—are increased after aneurysmal subarachnoid hemorrhage (SAH), but the functional significance remains undetermined. This study was conducted to evaluate whether modified citrus pectin (MCP; galectin-3 inhibitor) prevents post-SAH early brain injury, focusing on blood-brain barrier disruption. Methods— C57BL/6 male adult mice (n=251) underwent sham or filament perforation SAH modeling, followed by a random intracerebroventricular injection of vehicle or drug at 30 minutes post-modeling. First, vehicle-treated and 0.8, 4, 16, or 32 µg MCP-treated mice were assessed by neuroscore and brain water content at 24 and 48 hours post-modeling. Second, Evans blue extravasation, Western blotting, coimmunoprecipitation and immunostaining were performed in vehicle-treated or 4 µg MCP-treated mice at 24 hours post-modeling. Third, vehicle or R-galectin-3 (recombinant galectin-3) was administered to SAH mice simultaneously with vehicle or MCP, and neuroscore and Evans blue extravasation were evaluated at 24 hours post-modeling. Fourth, vehicle or R-galectin-3 was administered to MCP-treated SAH mice at 24 hours, and neuroscore and IgG immunostaining were evaluated at 48 hours post-SAH. Results— Among tested dosages, 4 µg MCP showed the best neuroprotective effects as to preventing neurological impairments and brain edema at 24 to 48 hours post-SAH. Four micrograms MCP attenuated post-SAH blood-brain barrier disruption and galectin-3 upregulation in brain capillary endothelial cells, associated with inactivation of ERK (extracellular signal-related kinase) 1/2, STAT (signal transducer and activator of transcription)-3, and MMP (matrix metalloproteinase)-9, and the consequent preservation of a tight junction protein ZO-1 (zonula occludens-1). Coimmunoprecipitation assay demonstrated physical interactions between galectin-3 and TLR (Toll-like receptor) 4. R-galectin-3 blocked the neuroprotective effects of MCP. Conclusions— MCP prevents post-SAH blood-brain barrier disruption possibly by inhibiting galectin-3, of which the mechanisms may include binding to TLR4 and activating ERK1/2, STAT-3, and MMP-9. This study suggests galectin-3 to be a novel therapeutic target against post-SAH early brain injury.

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.

Subarachnoid hemorrhage (SAH) is a devastating disease. Cerebral vasospasm is still an important cause of post-SAH poor outcomes, but its mechanisms remain unveiled. Activation of epidermal growth factor receptor (EGFR) is suggested to cause vasoconstriction in vitro, but no report has demonstrated the involvement of EGFR in vasospasm development after SAH in vivo. Cross-talk of EGFR and vascular endothelial growth factor (VEGF) receptor, which may affect post-SAH vasospasm, was also reported in cancer cells, but has not been demonstrated in post-SAH vasospasm. The aim of this study was to investigate whether EGFR as well as EGFR-VEGF receptor cross-talk engage in the development of cerebral vasospasm in a mouse SAH model. C57BL6 mice underwent endovascular perforation SAH or sham modeling. At 30 min post-modeling, mice were randomly administrated vehicle or 2 doses of selective EGFR inhibitors intracerebroventricularly. A higher dose of the inhibitor significantly prevented post-SAH neurological impairments at 72 h and vasospasm at 24 h associated with suppression of post-SAH activation of EGFR and extracellular signal-regulated kinase (ERK) 1/2 in the cerebral artery wall, especially in the smooth muscle cell layers. Anti-EGFR neutralizing antibody also showed similar effects. However, neither expression levels of VEGF nor activation levels of a major receptor of VEGF, VEGF receptor-2, were affected by SAH and two kinds of EGFR inactivation. Thus, this study first showed that EGFR-ERK1/2 pathways may be involved in post-SAH vasospasm development, and that EGFR-VEGF receptor cross-talk may not play a significant role in the development of vasospasm in mice.

The purpose of this study was to examine whether oral administration of an angiotensin II type 1 receptor blocker (ARB) inhibited in-stent neointimal hyperplasia in carotid arteries of hypercholesterolemic rabbits.Eleven male New Zealand white rabbits were subjected to endothelial injuries of the right common carotid arteries using a balloon catheter and then received chow containing 1% cholesterol for 6 weeks. A balloon-expandable stainless steel stent was subsequently inserted at the injured sites of the arteries. After stenting, five rabbits were randomly treated with an oral ARB, candesartan cilexetil (5 mg/kg per day orally), while the remaining six rabbits acted as untreated controls. Four weeks after the implantation, the rabbits were killed, followed by collection of the arteries including the stents. After careful removal of the stents, tissue sections were prepared and analyzed by morphometric and immunohistochemical methods.The mean thickness of the neointima was 53.6 ± 17.0 μm in the ARB-treated group, which was significantly reduced compared to 95.9 ± 16.7 μm in the control group (P = 0.0012). Immunohistochemistry showed a decrease in accumulation of macrophages and tenascin-C expression in the arterial wall in the ARB-treated animals.This study suggested that systemic administration of an ARB suppressed neointimal hyperplasia in the carotid artery following stent implantation by the anti-inflammatory effects, although the animal cohort tested was rather small. This finding implies that ARBs may be useful and practical agents for protection against in-stent restenosis in humans, and warrants further basic and clinical studies.

A matricellular protein tenascin-C (TNC) has been suggested to play a role in the pathogenesis of cerebral vasospasm after subarachnoid hemorrhage (SAH), but the direct evidence remains lacking. In this study, we examined effects of TNC knockout (TNKO) on cerebral vasospasm after experimental SAH in mice. C57BL/6 wild-type (WT) or TNKO mice were subjected to SAH by endovascular puncture. Ten WT and ten TNKO mice were randomized to WT sham (n = 4), TNKO sham (n = 4), WT SAH (n = 6), and TNKO SAH (n = 6) groups. In addition to neurobehavioral impairments and severity of SAH, cerebral vasospasm was assessed by morphometric measurements of the left internal carotid artery (ICA). Infiltration of inflammatory cells in the subarachnoid periarterial space was also assessed, and expressions of TNC and mitogen-activated protein kinases (MAPKs) in the ICA were immunohistochemically evaluated at 24 h post-surgery. TNC was induced in the smooth muscle cell layers and the adventitia in the spastic ICAs as well as the periarterial inflammatory cells in WT SAH mice. Compared with WT SAH mice, TNKO SAH mice showed better neurological scores and less severe cerebral vasospasm, as well as fewer inflammatory cell infiltration in the periarterial space. Post-SAH activation of MAPKs in the smooth muscle cell layers of the ICAs was also prevented in TNKO SAH mice. The findings in the present study suggest that TNC causes the development of cerebral vasospasm via pro-inflammatory effects and activation of MAPKs.

To elucidate the distribution of improved pain and numbness after cervical decompression surgery in patients with cervical spine disorders.This study included 4 men and 5 women aged 45 to 71 years(mean 58 years)presenting with radiculopathy and 50 men and 17 women aged 35 to 88 years(mean 66 years)presenting with myelopathy.All 9 patients with radiculopathy presented with neck pain, and 3 presented with cervical angina. Among the patients with myelopathy, 2 presented with headache, 2 with onion-skin facial pain, 29 with neck pain, 8 with truncal pain, 7 with low back pain, 4 with numbness below the T4 dermatomal area, 1 with penile pain, 61 with arm pain, 49 with leg pain, and 2 without pain or numbness. Patients with myelopathy presenting with preoperative neck and arm pain had significantly better recovery rates compared to patients without such pain.Patients with cervical spine disorders present with pain and numbness in various areas. Preoperative neck pain and arm pain are indicators for better recovery in patients with myelopathy.

Aneurysmal subarachnoid hemorrhage (aSAH) causes serious brain injury and the mechanisms have not been completely found out yet. The causative factors of brain injury initiated by aneurysm rupture, which is called as early brain injury (EBI), consists of elevated intracranial pressure, cerebral hypoperfusion and blood contents directly exposed to brain surface. At 4-14 days post-aSAH, delayed cerebral ischemia (DCI) often develops and may critically worsen neurological outcomes. DCI may be the sequence of EBI. Understanding the complex mechanisms of post-aSAH brain injury (EBI and DCI) is thus important to improve the outcome. In addition, many biomarkers possibly associated with EBI, DCI and neurological outcome have been investigated, but none of them is conclusive. A matricellular protein periostin is emerging as a potentially important contributor to EBI and DCI, and can serve as a biomarker and a therapeutic molecular target for EBI and DCI. In this article, the possible role of periostin in aSAH is reviewed.

OBJECTIVEChronic hydrocephalus develops in association with the induction of tenascin-C (TNC), a matricellular protein, after aneurysmal subarachnoid hemorrhage (SAH). The aim of this study was to examine if cilostazol, a selective inhibitor of phosphodiesterase Type III, suppresses the development of chronic hydrocephalus by inhibiting TNC induction in aneurysmal SAH patients.METHODSThe authors retrospectively reviewed the factors influencing the development of chronic shunt-dependent hydrocephalus in 87 patients with Fisher Grade 3 SAH using multivariate logistic regression analyses. Cilostazol (50 or 100 mg administered 2 or 3 times per day) was administered from the day following aneurysmal obliteration according to the preference of the attending neurosurgeon. As a separate study, the effects of different dosages of cilostazol on the serum TNC levels were chronologically examined from Days 1 to 12 in 38 SAH patients with Fisher Grade 3 SAH.RESULTSChronic hydrocephalus occurred in 12 of 36 (33.3%), 5 of 39 (12.8%), and 1 of 12 (8.3%) patients in the 0 mg/day, 100 to 200 mg/day, and 300 mg/day cilostazol groups, respectively. The multivariate analyses showed that older age (OR 1.10, 95% CI 1.13–1.24; p = 0.012), acute hydrocephalus (OR 23.28, 95% CI 1.75–729.83; p = 0.016), and cilostazol (OR 0.23, 95% CI 0.05–0.93; p = 0.038) independently affected the development of chronic hydrocephalus. Higher dosages of cilostazol more effectively suppressed the serum TNC levels through Days 1 to 12 post-SAH.CONCLUSIONSCilostazol may prevent the development of chronic hydrocephalus and reduce shunt surgery, possibly by the inhibition of TNC induction after SAH.

Toll-like receptor 4 (TLR4) signaling may play a crucial role in the occurrence of cerebral vasospasm after subarachnoid hemorrhage (SAH). The main purpose of this study was to assess if selective blockage of TLR4 on cerebral arteries prevents cerebral vasospasm development and neurological impairments after SAH in mice. One hundred fourteen mice underwent endovascular perforation SAH or sham operation and were randomly divided into the following 6 groups: sham+vehicle, sham+LPS-RS ultrapure 8 μg, sham+LPS-RS ultrapure 40 μg, SAH+vehicle, SAH+LPS-RS ultrapure 8 μg, and SAH+LPS-RS ultrapure 40 μg. A selective TLR4 antagonist, LPS-RS ultrapure (8 or 40 μg), was administered intracerebroventricularly to mice at 30 min, and the effects were evaluated by neurobehavioral tests and India-ink angiography at 24–48 h, and Western blotting and immunohistochemistry on cerebral arteries at 24 h post-SAH. Higher but not lower dosages of LPS-RS ultrapure significantly prevented post-SAH neurological impairments and ameliorated cerebral vasospasm. SAH caused TLR4 activation and cyclooxygenase-1 (COX1) upregulation in the endothelial cells and smooth muscle cells of spastic cerebral arteries, both of which were significantly suppressed by LPS-RS ultrapure. Another selective TLR4 antagonist, IAXO-102, which has a different binding site from LPS-RS ultrapure, also showed similar protective effects to LPS-RS ultrapure. These findings suggest that TLR4 signaling is implicated in cerebral vasospasm development at least partly via COX1 upregulation, and that TLR4 antagonists have therapeutic potential as a new therapy against cerebral vasospasm.

Vascular endothelial growth factor (VEGF) has been implicated in the pathogenesis of brain edema formation after experimental subarachnoid hemorrhage (SAH). In this study, we evaluated the effect of anti-VEGF antibody neutralization on brain edema formation after experimental SAH in mice. Mice underwent sham operation or filament puncture SAH and were assigned to sham, SAH + vehicle, or SAH + anti-VEGF antibody groups. Vehicle or anti-VEGF antibody was administrated by an intracerebroventricular injection at 30 min post-SAH. After 24 h of SAH modeling, neurological score was recorded to evaluate neurobehavioral functions, brain water content was calculated to assess the level of brain edema, and immunohistochemistry of immunoglobulin (Ig) G was performed to evaluate the permeability of the blood-brain barrier (BBB). Anti-VEGF antibody significantly ameliorated neurological score and brain edema after SAH compared with the SAH + vehicle group. Immunohistochemistry showed that post-SAH IgG extravasation in brain tissue was suppressed by anti-VEGF antibody. This study suggests that VEGF is involved in brain edema formation after SAH, and that anti-VEGF antibody can decrease BBB permeability, suppress brain edema formation, and improve functional outcome after 24 h of SAH.