Mutsumi Fujii

Isoflurane Attenuates Blood–Brain Barrier Disruption in Ipsilateral Hemisphere After Subarachnoid Hemorrhage in Mice

Background and Purpose— We examined effects of isoflurane, volatile anesthetics, on blood–brain barrier disruption in the endovascular perforation model of subarachnoid hemorrhage (SAH) in mice. Methods— Animals were assigned to sham-operated, SAH+vehicle–air, SAH+1%, or 2% isoflurane groups. Neurobehavioral function, brain water content, Evans blue dye extravasation, and Western blotting for sphingosine kinases, occludin, claudin-5, junctional adhesion molecule, and vascular endothelial cadherin were evaluated at 24 hours post-SAH. Effects of sphingosine kinase (N,N-dimethylsphingosine) or sphingosine-1-phosphate receptor-1/3 (S1P1/3) inhibitors (VPC23019) on isofluranes action were also examined. Results— SAH aggravated neurological scores, brain edema, and blood–brain barrier permeability, which were prevented by 2% but not 1% isoflurane posttreatment. Two percent isoflurane increased sphingosine kinase-1 expression and prevented a post-SAH decrease in expressions of the blood–brain barrier-related proteins. Both N,N-dimethylsphingosine and VPC23019 abolished the beneficial effects of isoflurane. Conclusions— Two percent isoflurane can suppress post-SAH blood–brain barrier disruption, which may be mediated by sphingosine kinase 1 expression and sphingosine-1-phosphate receptor-1/3 activation.

Hyperbaric oxygen preconditioning attenuates hyperglycemia-enhanced hemorrhagic transformation by inhibiting matrix metalloproteinases in focal cerebral ischemia in rats

Hyperglycemia dramatically aggravates brain infarct and hemorrhagic transformation (HT) after ischemic stroke. Oxidative stress and matrix metalloproteinases (MMPs) play an important role in the pathophysiology of HT. Hyperbaric oxygen preconditioning (HBO-PC) has been proved to decrease oxidative stress and has been demonstrated to be neuroprotective in experimental stroke models. The present study determined whether HBO-PC would ameliorate HT by a pre-ischemic increase of reactive oxygen species (ROS) generation, and a suppression of MMP-2 and MMP-9 in hyperglycemic middle cerebral artery occlusion (MCAO) rats. Rats were pretreated with HBO (100% O₂, 2.5 atmosphere absolutes) 1 h daily for 5 days before MCAO. Acute hyperglycemia was induced by an injection of 50% dextrose. Neurological deficits, infarction volume and hemorrhagic volume were assessed 24 h and 7 days after ischemia. ROS scavenger n-acetyl cysteine (NAC), hypoxia-inducible factor-1α (HIF-1α), inhibitor 2-methoxyestradiol (2ME2) and activator cobalt chloride (CoCl₂), and MMP inhibitor SB-3CT were administrated for mechanism study. The activity of MMP-2 and MMP-9, and the expression HIF-1α were measured. HBO-PC improved neurological deficits, and reduced hemorrhagic volume; the expression of HIF-1α was significantly decreased, and the activity of MMP-2 and MMP-9 was reduced by HBO-PC compared with vehicle group. Our results suggested that HBO-PC attenuated HT via decreasing HIF-1α and its downstream MMP-2 and MMP-9 in hyperglycemic MCAO rats.

Inhibition of Rho kinase by Hydroxyfasudil Attenuates Brain Edema after Subarachnoid Hemorrhage in Rats

The blood-brain barrier (BBB) disruption and brain edema are important pathophysiologies of early brain injury after subarachnoid hemorrhage (SAH). This study is to evaluate whether Rho kinase (Rock) enhances BBB permeability via disruption of tight junction proteins during early brain injury. Adult male rats were assigned to five groups; Sham-operated, SAH treated with saline, a Rock inhibitor hydroxyfasudil (HF) (10 mg/kg) treatment at 0.5 h after SAH, HF treatment at 0.5 and 6 h (10 mg/kg, each) after SAH, and another Rock inhibitor Y27632 (10 mg/kg) treatment at 0.5 h after SAH. The perforation model of SAH was performed and neurological score and brain water content were evaluated 24 and 72 h after surgery. Evans blue extravasation, Rock activity assay, and western blotting analyses were evaluated 24 h after surgery. Treatment of HF significantly improved neurological scores 24 h after SAH. Single treatment with HF and Y27632, and two treatments with HF reduced brain water content in the ipsilateral hemisphere. HF reduced Evans blue extravasation in the ipsilateral hemisphere after SAH. Rock activity increased 24 h after SAH, and HF reversed the activity. SAH significantly decreased the levels of tight junction proteins, occludin and zonula occludens-1 (ZO-1), and HF preserved the levels of occluding and ZO-1 in ipsilateral hemisphere. In conclusion, HF attenuated BBB permeability after SAH, possibly by protection of tight junction proteins.

Role of SCH79797 in Maintaining Vascular Integrity in Rat Model of Subarachnoid Hemorrhage

Background and Purpose— Plasma thrombin concentration is increased after subarachnoid hemorrhage (SAH). However, the role of thrombin receptor (protease-activated receptor-1 [PAR-1]) in endothelial barrier disruption has not been studied. The aims of this study were to investigate the role of PAR-1 in orchestrating vascular permeability and to assess the potential therapeutics of a PAR-1 antagonist, SCH79797, through maintaining vascular integrity. Methods— SCH79797 was injected intraperitoneally into male Sprauge-Dawley rats undergoing SAH by endovascular perforation. Assessment was conducted at 24 hours after SAH for brain water content, Evans blue content, and neurobehavioral testing. To explore the role of PAR-1 activation and the specific mechanism of SCH79797’s effect after SAH, Western blot, immunoprecipitation, and immunofluorescence of hippocampus tissue were performed. A p21-activated kinase-1 (PAK1) inhibitor, IPA-3, was used to explore the underlying protective mechanism of SCH79797. Results— At 24 hours after SAH, animals treated with SCH79797 demonstrated a reduction in brain water content, Evans blue content, and neurobehavioral deficits. SCH79797 also attenuated PAR-1 expression and maintained the level of vascular endothelial-cadherin, an important component of adherens junctions. Downstream to PAR-1, c-Src–dependent activation of p21-activated kinase-1 led to an increased serine/threonine phosphorylation of vascular endothelial-cadherin; immunoprecipitation results revealed an enhanced binding of phosphorylated vascular endothelial-cadherin with endocytosis orchestrator &bgr;-arrestin-2. These pathological states were suppressed after SCH79797 treatment. Conclusions— PAR-1 activation after SAH increases microvascular permeability, at least, partly through a PAR-1-c-Src-p21-activated kinase-1-vascular endothelial-cadherin phosphorylation pathway. Through suppressing PAR-1 activity, SCH79797 plays a protective role in maintaining microvascular integrity after SAH.

Hyperbaric oxygen preconditioning attenuates hyperglycemia enhanced hemorrhagic transformation after transient MCAO in rats.

BackgroundHemorrhagic transformation (HT) can be a devastating complication of ischemic stroke. Hyperbaric oxygen preconditioning (HBO-PC) has been shown to improve blood-brain barrier (BBB) permeability in stroke models. The purpose of this study is to examine whether HBO-PC attenuates HT after focal cerebral ischemia, and to investigate whether the mechanism of HBO-PC against HT includes up-regulation of antioxidants in hyperglycemic rats.MethodsMale Sprague-Dawley rats (280-320 g) were divided into the following groups: sham, middle cerebral artery occlusion (MCAO) for 2 h, and MCAO treated with HBO-PC. HBO-PC was conducted giving 100% oxygen at 2.5 atm absolute (ATA), for 1 h at every 24 h interval for 5 days. At 24 h after the last session of HBO-PC, rats received an injection of 50% glucose (6 ml/kg intraperitoneally) and were subjected to MCAO 15 min later. At 24 h after MCAO, neurological behavior tests, infarct volume, blood-brain barrier permeability, and hemoglobin content were measured to evaluate the effect of HBO-PC. Western blot analysis of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) was evaluated at multiple time-points before and after MCAO.ResultsHBO-PC improved neurological behavior test, and reduced infarction volume, HT and Evans blue extravasation in the ipsilateral hemisphere at 24 h after MCAO. Western blot analysis failed to demonstrate up-regulation of Nrf2 in HBO-PC group before and after MCAO. Paradoxically, HBO-PC decreased HO-1 expression at 24 h after MCAO, as compared with htMCAO group.ConclusionsHBO-PC improved neurological deficits, infarction volume, BBB disruption, and HT after focal cerebral ischemia. However, its mechanism against focal cerebral ischemia and HT may not include activation of Nrf2 and subsequent HO-1 expression.

Cannabinoid type 2 receptor stimulation attenuates brain edema by reducing cerebral leukocyte infiltration following subarachnoid hemorrhage in rats.

Early brain injury (EBI), following subarachnoid hemorrhage (SAH), comprises blood-brain barrier (BBB) disruption and consequent edema formation. Peripheral leukocytes can infiltrate the injured brain, thereby aggravating BBB leakage and neuroinflammation. Thus, anti-inflammatory pharmacotherapies may ameliorate EBI and provide neuroprotection after SAH. Cannabinoid type 2 receptor (CB2R) agonism has been shown to reduce neuroinflammation; however, the precise protective mechanisms remain to be elucidated. This study aimed to evaluate whether the selective CB2R agonist, JWH133 can ameliorate EBI by reducing brain-infiltrated leukocytes after SAH. Adult male Sprague-Dawley rats were randomly assigned to the following groups: sham-operated, SAH with vehicle, SAH with JWH133 (1.0mg/kg), or SAH with a co-administration of JWH133 and selective CB2R antagonist SR144528 (3.0mg/kg). SAH was induced by endovascular perforation, and JWH133 was administered 1h after surgery. Neurological deficits, brain water content, Evans blue dye extravasation, and Western blot assays were evaluated at 24h after surgery. JWH133 improved neurological scores and reduced brain water content; however, SR144528 reversed these treatment effects. JWH133 reduced Evans blue dye extravasation after SAH. Furthermore, JWH133 treatment significantly increased TGF-β1 expression and prevented an SAH-induced increase in E-selectin and myeloperoxidase. Lastly, SAH resulted in a decreased expression of the tight junction protein zonula occludens-1 (ZO-1); however, JWH133 treatment increased the ZO-1 expression. We suggest that CB2R stimulation attenuates neurological outcome and brain edema, by suppressing leukocyte infiltration into the brain through TGF-β1 up-regulation and E-selectin reduction, resulting in protection of the BBB after SAH.

Cannabinoid Receptor Type 2 Agonist Attenuates Acute Neurogenic Pulmonary Edema by Preventing Neutrophil Migration after Subarachnoid Hemorrhage in Rats.

We evaluated whether JWH133, a selective cannabinoid type 2 receptor (CB2R) agonist, prevented neurogenic pulmonary edema (NPE) after subarachnoid hemorrhage (SAH) by attenuating inflammation. Adult male rats were assigned to six groups: sham-operated, SAH with vehicle, SAH with JWH133 (0.3, 1.0, or 3.0 mg/kg) treatment 1 h after surgery, and SAH with JWH133 (1.0 mg/kg) at 1 h with a selective CB2R antagonist, SR144528 (3.0 mg/kg). The perforation model of SAH was performed and pulmonary wet-to-dry weight ratio was evaluated 24 and 72 h after surgery. Western blot analyses and immunohistochemistry were evaluated 24 h after surgery. JWH133 (1.0 mg/kg) significantly and most strongly improved lung edema 24 h after SAH. SR144528 administration significantly reversed the effects of JWH133 (1.0 mg/kg). SAH-induced increasing levels of myeloperoxidase (MPO) and decreasing levels of a tight junction (TJ) protein, junctional adhesion molecule (JAM)-A, were ameliorated by JWH133 (1.0 mg/kg) administration 24 h after SAH. Immunohistochemical assessment also confirmed substantial leukocyte infiltration in the outside of vessels in SAH, which were attenuated by JWH133 (1.0 mg/kg) injection. CB2R agonist ameliorated lung permeability by inhibiting leukocyte trafficking and protecting tight junction proteins in the lung of NPE after SAH.

Inflammation as a Therapeutic Target after Subarachnoid Hemorrhage: Advances and Challenges

Subarachnoid hemorrhage (SAH) results from the rupture of an intracranial aneurysm, and the first consequent events are increased intracranial pressure (ICP), reduced cerebral perfusion pressure (CPP), and decreased cerebral blood flow (CBF). The resultant hypoxic state alters autoregulation, ionic homeostasis, and excitotoxicity as well as initiates secondary injuries such as cytotoxic edema, blood-brain barrier (BBB) disruption, inflammation, and apoptotic cell death. Inflammation persists through hemorrhage degradation in the subarachnoid space. Several different aspects of the inflammatory response have been demonstrated in stroke pathogenesis, including cellular response (e.g., leukocyte adherence and microglia activation), expression of adhesion molecules (e.g., selectins, integrins, and immunoglobulin superfamily), production of inflammatory mediators (e.g., cytokines, nitric oxide/nitric oxide synthase (NO/NOS), and free radicals), and accumulation of platelet aggregates. Since all of these inflammatory aspects lead to brain edema and cell death, inflammation could be a particularly important target for designing therapeutic strategies against secondary injuries after SAH. Given these inflammatory contributions could be seen in large vessels, a plethora of research has been intended to reduce cerebral vasospasm (CVS) after SAH. The main research field, however, is moving toward studying early brain injury (EBI) because some human research demonstrated the morphological alleviation of CVS alone might not improve the functional recovery in patients after SAH. This chapter provides the current knowledge of the inflammatory response, translational research, and human clinical trials in SAH as well as discusses emerging opportunities for novel therapeutic strategies for clinical management of SAH.

SAH Models: Review, New Modification, and Prospective

Subarachnoid hemorrhage (SAH) is a devastating type of hemorrhagic stroke. It is characterized by spontaneous or traumatic bleeding in the subarachnoid space and is associated with a high rate of morbidity and mortality. A reproducible animal model of SAH that mimics the acute and delayed brain injury history after SAH will be an invaluable tool for exploring the underlying mechanisms of SAH-induced brain injury and evaluating potential therapeutic interventions. At present, a number of models have been developed, mainly the double injection model and the endovascular puncture model. While different species have been studied, rodents have become the most popular and widely utilized animal subjects. In this summary, we will explore in detail the various models and animal species. We will also introduce the emerging modified model, which was recently developed within the past 5 years, and discuss the prospective study.

Subarachnoid Hemorrhage-Triggered Acute Hypotension Is Associated with Left Ventricular Cardiomyocyte Apoptosis in a Rat Model.

Whether hypotension that occurs due to neurogenic stunned myocardium after subarachnoid hemorrhage (SAH) is associated with cardiomyocyte apoptotic cell death remains unknown. In this study, 18 male rats were subjected to sham or the endovascular perforation model of SAH surgery. Based on the mean arterial pressure (MAP) after SAH, rats were separated into SAH with hypotension (SAH hypotension) or SAH with blood pressure preservation (SAH BP preservation) groups. All animals were euthanized 2 h after the surgical procedure. Hearts were removed and separated transversely into base and apex parts, then Western blot analyses and immunohistochemistry were performed only in the apex part. One rat died as a result of severe SAH and two rats with mild SAH were excluded. We analyzed data from 15 rats that were divided into three groups: sham, SAH hypotension, and SAH BP preservation (n = 5, each). There was a significantly higher cleaved caspase-3/caspase-3 ratio in the SAH hypotension group compared with sham and the SAH BP preservation group. Cardiomyocyte apoptosis was demonstrated in the SAH rats. This is the first experimental report that describes SAH-induced neurogenic stunned myocardium with ensuing hypotension may result from the acute apoptotic cardiomyocyte cell death in the left ventricle.